Polypeptides, nucleic acids and uses thereof

ABSTRACT

We describe an ELABELA polypeptide comprising a sequence CXXXRCXXXHSRVPFP (SEQ ID NO: 1), in which X signifies an amino acid residue, such as a sequence selected from the group consisting of: SEQ ID NO: 2 to SEQ ID NO: 18, preferably CLQRRCMPLHSRVPFP (SEQ ID NO: 2), or a fragment, homologue, variant or derivative thereof, which polypeptide is capable of maintaining self-renewal and/or pluripotency of a stem cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §120 of U.S. Ser. No.14/496,600, filed on Sep. 25, 2014, which claims benefit under 35 U.S.C.§119(e) of U.S. Provisional Application Ser. No. 61/911,276 filed Dec.3, 2013, the contents of which are herein incorporated by reference intheir entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 12, 2014, isnamed 049595-080181-US_SL.txt and is 67,735 bytes in size.

FIELD

The present invention relates to the fields of medicine, cell biology,molecular biology and genetics. This invention relates to the field ofmedicine.

BACKGROUND

Hormonal peptides are an important class of secreted signalingmolecules. Endogenous peptides are most notable for their functions ininnate defense as antimicrobial peptides (Cederlund et al., 2011), inimmune regulation as chemokines (Bonecchi et al., 2009) and inmodulation of behavior as neuropeptides (van den Pol, 2012).Deficiencies in hormonal peptides are the cause of several humandiseases, the most prominent being the loss of INSULIN or INSULINresistance in diabetes mellitus. Deficiency in the neuropeptideHYPOCRETIN causes narcolepsy (Nishino et al., 2000; Peyron et al., 2000)while anomalies in the regulation of the appetite and satiety hormonesLEPTIN (Montague et al., 1997) and GHRELIN are the underlying causes forcongenital obesity and hyperphagia in Prader-Willi syndrome (Cummings etal., 2002).

The discovery of new peptide-encoding genes is challenging, since theiropen reading frames (ORFs) are small and often overlooked by size-biasedORF prediction algorithms. This limitation has lead to theirunder-prediction or classification as non-coding transcripts (Frith etal., 2006). Equally challenging is the matching of these hormones totheir cognate cell surface receptors. The vast majority of smallsignaling peptides are known to bind and signal through G-coupledprotein receptors (GPCRs), the largest family of cell surface receptors(Rasmussen et al., 2007). GPCRs can be broadly classified into twocategories based on the source of their ligands (Vassilatis et al.,2003). Chemosensory-GPCRs sense environmental cues such as odorants,tastants and pheromones, while endo-GPCRs transduce signals originatingfrom endogenous compounds such as peptide hormones, amines, nucleosidesor lipids. Recent studies estimate the number of endo-GPCRs in the humangenome to be close to 370 (Vassilatis et al., 2003). Approximately 140of these (40%) have no known ligands and are therefore orphaned GPCRreceptors. As such, the discovery and pairing of novel endogenoushormones to their cognate receptors remains a considerable endeavour.

Whereas many peptide hormones have been characterized and shown to playkey roles in adult physiology, an involvement for these tiny signalingmolecules during early development has not been established. Duringembryogenesis, six key signaling pathways namely WNT, BMP/NODAL,FGF/IGF, NOTCH, HEDGEHOG and HIPPO are crucial for embryonic patterning.In particular IGF/FGF and NODAL are essential for maintainingpluripotency in human embryonic stem cells (hESCs) (Dalton, 2013). Asidefrom INSULIN/IGF, FGF and TGFβ/ACTIVIN/NODAL, no other soluble factorshave been isolated from feeder or hESC-conditioned media and proven tobe necessary for hESC culture (Hughes et al., 2011).

To our knowledge, no hormonal peptide has ever been implicated inmaintaining the self-renewal capacity of hESCs or their ability todifferentiate into any of the three embryonic germ layers.

SUMMARY

According to a 1^(st) aspect of the present invention, we provide anELABELA polypeptide. The ELABELA polypeptide may comprise a sequenceCXXXRCXXXHSRVPFP (SEQ ID NO: 1). X may signify an amino acid residue.

We also provide a fragment, homologue, variant or derivative of such apolypeptide.

The polypeptide may have an activity, such as a biological activity ofan ELABELA polypeptide. For example, the polypeptide may be capable ofmaintaining self-renewal of a stem cell. It may be capable ofmaintaining pluripotency of a stem cell. It may be capable of doingboth.

The ELABELA polypeptide may comprise a basic residue at or aboutposition −7 upstream of the sequence CXXXRCXXXHSRVPFP (SEQ ID NO: 1).

The ELABELA polypeptide may comprise a basic residue at or aboutposition −8 upstream of the sequence CXXXRCXXXHSRVPFP (SEQ ID NO: 1).

The basic residue may comprise K (lysine). It may comprise R (arginine).

The ELABELA polypeptide may comprise a pair of basic residues at orabout positions −7 and −8 upstream of the sequence CXXXRCXXXHSRVPFP (SEQID NO: 1). The pair of basic residues may comprise KK. They may compriseKR. They may comprise RK. They may comprise RR.

The ELABELA polypeptide may comprise a sequence selected from the groupconsisting of: SEQ ID NO: 2 to SEQ ID NO: 18. The ELABELA polypeptidemay comprise a human ELABELA sequence shown as SEQ ID NO: 2.

The ELABELA polypeptide may further comprise a signal sequence. Thesignal sequence may comprise a human ELABELA signal sequence such asshown in SEQ ID NO: 19.

The ELABELA polypeptide may comprise a sequence selected from the groupconsisting of: SEQ ID NO: 20 to SEQ ID NO: 36. The ELABELA polypeptidemay comprise a human ELABELA sequence shown as SEQ ID NO: 20.

The ELABELA polypeptide may comprise an intramolecular covalent bondbetween the cysteine residues at positions 1 and 6, with reference tothe numbering in the sequence CXXXRCXXXHSRVPFP (SEQ ID NO: 1). TheELABELA polypeptide may be such that one or both cysteine residuescomprise a reduced cysteine, for example having a sulfhydryl group.

The ELABELA polypeptide may comprise a mutation of the residue atposition 31. The ELABELA polypeptide may comprise a mutation of theresidue at position 32. The ELABELA polypeptide may comprise a R31Gsubstitution. The ELABELA polypeptide may comprise a R31A substitution.The ELABELA polypeptide may comprise a K31G substitution. The ELABELApolypeptide may comprise a K31A substitution. The ELABELA polypeptidemay comprise an R32G substitution. The ELABELA polypeptide may comprisean R32A substitution. The ELABELA polypeptide may comprise a K32Gsubstitution. The ELABELA polypeptide may comprise a K32A substitution.The residue numbering may be by reference to the position numbering of ahuman ELABELA sequence shown as SEQ ID NO: 20.

There is provided, according to a 2^(nd) aspect of the presentinvention, a nucleic acid comprising a sequence capable of encoding anELABELA polypeptide as set out above.

The ELABELA nucleic acid may comprise a nucleic acid sequence shown inany of SEQ ID NO. 37 to SEQ ID NO: 46. The ELABELA nucleic acid maycomprise a human ELABELA nucleic acid sequence SEQ ID NO: 37 or SEQ IDNO: 42.

We provide, according to a 3^(rd) aspect of the present invention,vector such as an expression vector comprising such a nucleic acidaccording. We further provide a host cell such as a bacterial, fungal oryeast cell comprising a such a vector or such a nucleic acid. We furtherprovide a transgenic non-human animal comprising such a host cell, sucha vector or such a nucleic acid. The transgenic non-human animal maycomprise a mammal The transgenic non-human animal may comprise a mouse.

As a 4^(th) aspect of the present invention, there is provided anantibody. The antibody may be capable of specifically binding to apolypeptide comprising the sequence CMPLHSRVPFP (SEQ ID NO: 52). Theantibody may be capable of specifically binding to a polypeptidecomprising the sequence QRPVNLTMRRKLRKHNC (SEQ ID NO: 53). The antibodymay be capable of specifically binding to a polypeptide comprising thesequence QRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP (SEQ ID NO: 2). The antibodymay be capable of specifically binding to an ELABELA polypeptide as setout above. The antibody may be capable of specifically binding to anELABELA polypeptide encoded by a nucleic acid as set out above. Whereantibodies that bind them are provided, it should be understood thatthese antigenic polypeptide fragments can also be useful and areprovided in their own right.

We provide, according to a 5^(th) aspect of the present invention, anshRNA or siRNA molecule capable of modulating any combination of theexpression, amount or activity of an ELABELA polypeptide set out above.The shRNA or siRNA may comprise a sequence selected from the groupconsisting of: SEQ ID NO: 47 to SEQ ID NO: 51.

As a 6^(th) aspect of the present invention, there is provided a methodof assaying a compound of interest. The method may comprise contactingan ELABELA polypeptide as set out above with a candidate compound andperforming an assay to determine if the candidate compound binds to theELABELA polypeptide. The method may comprise contacting an ELABELApolypeptide as set out above with a candidate compound and performing anassay to determine if the candidate compound modulates an activity ofthe ELABELA polypeptide. The assay may comprise contacting a cellexpressing an ELABELA polypeptide as set out above with a candidatecompound and performing an assay to determine if the candidate compoundcauses an elevated or reduced expression, amount or activity of theELABELA polypeptide in or of the cell. The method may further compriseisolating or synthesising the compound of interest so identified.

We provide, according to a 7^(th) aspect of the present invention, acompound of interest identified, isolated or synthesised by a method asset out above.

The present invention, in a 8^(th) aspect, provides a method ofdown-regulating any combination of the expression, amount or activity ofan ELABELA polypeptide. The method may comprise exposing the ELABELApolypeptide to an antibody as described. The method may compriseexposing the ELABELA polypeptide to an shRNA or siRNA as described. Themethod may comprise exposing the ELABELA polypeptide to a compound ofinterest as described.

The present invention, in a 9^(th) aspect, provides use of an ELABELApolypeptide as described, a nucleic acid as described, a vector, hostcell or transgenic non-human animal as described, an antibody asdescribed, an shRNA or siRNA molecule as described or a compound ofinterest as described in the treatment, prophylaxis or alleviation of anELABELA associated condition.

The ELABELA associated condition may comprise cardiac dysfunction,hypertension, or a cardiovascular anomaly in blood pressure, cardiaccontractility or fluid balance.

The ELABELA associated condition may comprise a cardiovascular diseasesuch as cardiac hypertrophy, coronary artery disease (CAD),atherosclerosis, post-infarct treatment, myocardial ischemia-reperfusioninjury or atrial fibrillation, coronary heart disease, heart failure,pulmonary arterial hypertension (PAH).

The ELABELA associated condition may comprise a condition associatedwith high blood pressure, such as hypertension, angina, congestive heartfailure or erectile dysfunction.

The ELABELA associated condition may comprise a condition associatedwith HIV infection, such as AIDS in an individual.

In a 10^(th) aspect of the present invention, there is provided use ofan ELABELA polypeptide as described. We further provide use of anELABELA polypeptide as described as a vasodilator.

According to an 11^(th) aspect of the present invention, we provide anELABELA polypeptide as described, a nucleic acid as described, a vector,host cell or transgenic non-human animal as described, an antibody asdescribed, an shRNA or siRNA molecule as described or a compound ofinterest as described for use in the treatment, prophylaxis oralleviation of an ELABELA associated condition, or for use as avasodilator.

We provide, according to a 12^(th) aspect of the invention, a method ofmanipulating a cell, the method comprising modulating, preferablyup-regulating, any combination of the expression, amount or activity ofan ELABELA polypeptide as described in or of the cell, such as byexposing the cell to an ELABELA polypeptide as described or introducinga nucleic acid as described or a vector as described into the cell.

There is provided, in accordance with a 13^(th) aspect of the presentinvention, a method of maintaining or enhancing self-renewal and/orpluripotency in or of a stem cell, the method comprising manipulating astem cell by a method as set out above.

The cell may comprise a stem cell. Up-regulation of the expression,amount or activity of an ELABELA polypeptide may result in maintenanceor enhancement of self-renewal and/or pluripotency in or of the stemcell.

As an 14^(th) aspect of the invention, we provide a method comprisingdetecting any combination of the expression, amount or activity of anELABELA polypeptide in or of a cell, such as a stem cell, tissue, organor organism.

We provide, according to a 15^(th) aspect of the invention, there isprovided a combination comprising a first part comprising a sequenceMPLHSRVPFP (SEQ ID NO: 54) or QRPVNLTMRRKLRKHN (SEQ ID NO: 55) or bothand a second part comprising a polypeptide of interest.

The combination may be such that the first part and the second part arecovalently joined, such as by chemical conjugation. The combination maycomprise a fusion protein comprising the first part and the second part.

According to a 16^(th) aspect of the present invention, we provide anexpression construct capable of expressing such a fusion protein.

There is provided, according to a 17^(th) aspect of the presentinvention, a method of detecting or quantifying a polypeptide ofinterest, the method comprising producing a combination as described anddetecting the presence of or quantifying the first part of thecombination.

We provide, according to a 18^(th) aspect of the present invention, amethod of isolating a polypeptide of interest. The method may compriseproducing a combination as described and exposing the combination to amolecule capable of specifically binding to the first part. The moleculecapable of specifically binding to the first part may comprise anantibody as set out above.

There is provided, according to a 19^(th) aspect of the presentinvention, a method comprising conjugating or otherwise joining apolypeptide of interest to a sequence MPLHSRVPFP (SEQ ID NO: 54) orQRPVNLTMRRKLRKHN (SEQ ID NO: 55) or both.

Also provided herein in some aspects are ELABELA polypeptide fragmentscomprising a sequence CXXXRCXXXHSRVPFP (SEQ ID NO: 1), where X is an/anyamino acid residue, and where the polypeptide fragment maintainsself-renewal, pluripotency, or both of a stem cell.

In some embodiments of these aspects and all such aspects describedherein, the fragment does not comprise a sequence of SEQ ID NOs: 60-76.

In some embodiments of these aspects and all such aspects describedherein, (a) an intramolecular covalent bond is present between thecysteine residues at positions 1 and 6 of SEQ ID NO: 1, or (b) one orboth cysteine residues at positions 1 and 6 of SEQ ID NO: 1 comprise areduced cysteine having a sulfhydryl group.

In some embodiments of these aspects and all such aspects describedherein, the ELABELA polypeptide fragment further comprises a label. Insome embodiments of these aspects and all such aspects described herein,wherein the label is a radioisotope. In some embodiments of theseaspects and all such aspects described herein, the radioisotope is ¹²⁵I.

In some embodiments of these aspects and all such aspects describedherein, the polypeptide fragment is derivatized.

In some embodiments of these aspects and all such aspects describedherein, the ELABELA polypeptide fragment further comprises a signalsequence. In some embodiments of these aspects and all such aspectsdescribed herein, the signal sequence comprises SEQ ID NO: 19.

In some embodiments of these aspects and all such aspects describedherein, the fragment further comprises seven additional amino acids atthe N-terminus of SEQ ID NO: 1, the ELABELA polypeptide fragment havinga sequence of SEQ ID NO: 162 (XXXXXXXCXXXRCXXXHSRVPFP), where position 1of SEQ ID NO: 162 is a basic amino acid residue, where the X atpositions 2-6, 8-10, and 13-15 is an/any amino acid residue, and wherethe polypeptide fragment maintains self-renewal, pluripotency, or bothof a stem cell.

In some embodiments of these aspects and all such aspects describedherein, the fragment does not comprise a sequence of SEQ ID NOs:181-197.

In some embodiments of these aspects and all such aspects describedherein, the basic residue at the position 1 is selected from K or R.

In some embodiments of these aspects and all such aspects describedherein, the fragment further comprises eight additional amino acids atthe N-terminus of SEQ ID NO: 1, said ELABELA polypeptide fragment havinga sequence of SEQ ID NO: 163 (XXXXXXXXCXXXRCXXXHSRVPFP), where position1 of SEQ ID NO: 163 is a basic amino acid residue, where the X atpositions 2-7, 9-11, and 14-17 is an/any amino acid residue, and wherethe polypeptide fragment maintains self-renewal, pluripotency, or bothof a stem cell.

In some embodiments of these aspects and all such aspects describedherein, the fragment does not comprise a sequence of SEQ ID NOs:164-180.

In some embodiments of these aspects and all such aspects describedherein, the basic residue at the position 1 is selected from K or R.

In some embodiments of these aspects and all such aspects describedherein, the fragment further comprises eight additional amino acids atthe N-terminus of SEQ ID NO: 1, said ELABELA polypeptide fragment havinga sequence of SEQ ID NO: 163 (XXXXXXXXCXXXRCXXXHSRVPFP), whereinpositions 1 and 2 of SEQ ID NO: 163 are a pair of basic amino acidresidues, wherein the X at positions 3-7, 10-12, and 15-17 is an/anyamino acid residue, and wherein the polypeptide fragment maintainsself-renewal, pluripotency, or both of a stem cell.

In some embodiments of these aspects and all such aspects describedherein, the fragment does not comprise a sequence of SEQ ID NOs:164-180.

In some embodiments of these aspects and all such aspects describedherein, the pair of basic residues at positions 1 and 2 is selected fromKK, KR, RK, and RR.

Also provided herein, in some aspects, are methods of making an ELABELApolypeptide or fragment thereof, the methods comprising:

(a) expressing a nucleic acid encoding a sequence comprisingCXXXRCXXXHSRVPFP (SEQ ID NO: 1) in a cell, wherein X is an/any aminoacid residue, and wherein the polypeptide fragment maintainsself-renewal, pluripotency, or both of a stem cell; or(b) using chemical synthesis to generate a synthetic polypeptide orfragment thereof comprising CXXXRCXXXHSRVPFP (SEQ ID NO: 1) or afragment having the sequence of SEQ ID NO: 53, wherein X is an/any aminoacid residue, and wherein the polypeptide fragment maintainsself-renewal, pluripotency, or both of a stem cell.

In some embodiments of these aspects and all such aspects describedherein, the cell expressing the nucleic acid encoding a sequencecomprising CXXXRCXXXHSRVPFP (SEQ ID NO: 1) is a bacterial, fungal, oryeast cell.

In some embodiments of these aspects and all such aspects describedherein, the sequence comprising CXXXRCXXXHSRVPFP (SEQ ID NO: 1) isselected from the group consisting of SEQ ID NOs: 2-36.

In some embodiments of these aspects and all such aspects describedherein, the ELABELA polypeptide or fragment thereof further comprises alabel. In some embodiments of these aspects and all such aspectsdescribed herein, the label is a radioisotope. In some embodiments ofthese aspects and all such aspects described herein, the radioisotope is¹²⁵I.

In some embodiments of these aspects and all such aspects describedherein, the polypeptide or fragment thereof is derivatized.

In some aspects, provided herein are isolated antibodies orantigen-binding fragments thereof that specifically bind to one or moreof the following:

-   -   (a) a polypeptide comprising the sequence CMPLHSRVPFP (SEQ ID        NO: 52);    -   (b) a polypeptide comprising the sequence QRPVNLTMRRKLRKHNC (SEQ        ID NO: 53);    -   (c) a polypeptide comprising the sequence        QRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP (SEQ ID NO: 2); and    -   (d) an ELABELA polypeptide comprising the sequence of any of SEQ        ID NOs: 1-36.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibody or antigen-binding fragment thereoffurther comprises a label.

Also provided herein, in some aspects, are immunoassay kits formeasuring or detecting ELABELA expression, the immunoassay kitscomprising:

-   -   (a) a coating antigen comprising one or more isolated antibodies        or antigen-binding fragments thereof that specifically binds to        one or more of the following:        -   (i) a polypeptide comprising the sequence CMPLHSRVPFP (SEQ            ID NO: 52);        -   (ii) a polypeptide comprising the sequence QRPVNLTMRRKLRKHNC            (SEQ ID NO: 53);        -   (iii) a polypeptide comprising the sequence            QRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP (SEQ ID NO: 2); or        -   (iv) an ELABELA polypeptide comprising the sequence of any            of SEQ ID NOs: 1-36; and    -   (b) instructions for using said coating antigen.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibodies or antigen-binding fragments thereof arelabelled.

In some embodiments of these aspects and all such aspects describedherein, the kit further comprises an enzyme labelled reagent, asecondary antibody that specifically binds to the isolated antibodies orantigen-binding fragments, a solid substrate, or any combinationthereof.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E. Belonging to the Core Embryonic Pluripotency, ELA Encodes aConserved Hormone.

FIG. 1A. NANOG, POU5F1 and PRDM14 syn-expression groups share a commonlist of 33 genes which define a core human pluripotency network. ELA isone of these genes.

FIG. 1B. ELA, like POU5F1, is rapidly silenced in hESCs during embryoidbody differentiation.

FIG. 1C. ELA encodes a conserved vertebrate protein of 54 amino-acidsconsisting of a secretory signal and a mature 32 amino-acid peptide. Thecarboxy terminus is invariant. White arrowhead: predicted signal peptidecleavage site between G22 and Q33. Double black arrowheads: possibleFURIN cleavage sites after conserved di-arginines R31R32 and R42R43motifs. N- and C-terminal epitopes chosen for α N and α C antibodyproduction are noted. The α C antibody is designed to recognize all ELApeptides regardless of species. FIG. 1C discloses SEQ ID NOs: 20, 22,25-28, and 30-33, respectively, in order of appearance.

FIG. 1D. Western blot for ELA which is translated, processed, secretedand recognized by the α C antibody as a 4 kDa single band whenover-expressed in Xenopus laevis embryos. Brefeldin A-mediatedinhibition of secretion blocks ELA processing.

FIG. 1E. The α C antibody recognizes both full-length ELA and processedELA whereas the α N antibody is specific to the mature processed ELApeptide.

FIGS. 2A-2H. ELA is an Endogenous Hormone Secreted by hESCs andEssential for their Survival.

FIG. 2A. By immunofluorescence in hESCs, endogenous ELA marked with theα C antibody co-localizes with the trans-Golgi network marked by TGN46.

FIG. 2B. Soluble extracellular ELA in conditioned medium of hESCs can bedepleted by siRNA mediated silencing and detected by a sandwich ELISAassay.

FIG. 2C. Over a period of 5 days, secreted ELA reaches nM concentrationsin the supernatant of hESCs cultures as measured by ELISA. Over the sameperiod, Dox-inducible shELA knockdown achieves nearly 85% depletion ofextracellular ELA.

FIG. 2D. As judged by immunofluorescence shELA, but not shβ2M, causeshESCS colonies to gradually differentiate and lose pluripotency markersPOU5F1, SSEA-3 and TRA-1-60.

FIG. 2E. Measured on the xCELLigence platform, shELA, but not shβ2M(inset) hESCs seeded as single cells exhibit significant growthimpairment.

FIG. 2F. An average shELA hESCS colonies are twice as small relative tocontrol hESCs.

FIG. 2G. shELA hESCs do not display overt changes in cell cycleprogression following release from a double thymidine block at G1/S.

FIG. 2H. By FACs analysis, shELA, but not shβ2M, causes hESCS grown insingle cells to undergo rapid apoptosis as judged by increased ANNEXIN Vand activated CASPASE 3.

FIGS. 3A-3K. Recombinant ELA Promotes hESCs Growth and Primes CellsTowards Mesendoderm Lineages.

FIG. 3A. Recombinant synthetically-produced ELA, and mutant ELA^(RR>GG)(R31 G, R32G), with free termini and intramolecular cystine bond betweenconserved C39 and C44 residues.

FIG. 3B. By immunofluorescence, recombinant ELA, but not ELA^(RR>GG),labeled with FITC is rapidly up-taken by hESCs.

FIG. 3C. By cell numbers, addition of exogenous ELA elicits increasedgrowth of hESCs while shELA hESCs show reduced growth relative tountreated hESCs.

FIG. 3D. Relative to untreated hESCs, addition of exogenous ELA, but notmutant ELA^(RR>GG), elicits increased survival of hESCs.

FIG. 3E. By real-time cell index analysis, supply of exogenous ELA, butnot mutant ELA^(RR>GG), affords increased growth of hESCs.

FIG. 3F. Exogenous ELA, but not mutant ELA^(RR>GG), is sufficient torescue the growth of shELA hESCs.

FIG. 3G. Affinity purified α C antibodies added to the medium of hESCsare sufficient to inhibit the growth of hESCs, demonstrating potentneutralizing activity.

FIG. 3H. The pro-growth and survival properties of recombinant ELA arespecific to hESCs culture and are not seen on multipotent hECs, orunipotent human chondrosarcoma and primary fibroblast cells.

FIG. 3I. By qPCR, hESCs treated with recombinant ELA expresssignificantly higher levels of mesendodermal markers. Conversely shELAhESCs have reduced levels of these same mesendodermal markers.

FIG. 3J. As judged by FACs, hESCs treated with recombinant ELA do notlose the stem cells cell surface markers SSEA-3 and TRA-1-60.

FIG. 3K. hESCs treated with recombinant ELA are not committed tomesendodermal lineages and can differentiate into all three germ layersupon embryoid differentiation as judged by qPCR analysis.

FIGS. 4A-4F. Generation of an Allelic Series of Mutant ela Zebrafish

FIG. 4A. By whole mount in situ hybridization in zebrafish embryos, elais found to be zygotically expressed and ubiquitous in the blastoderm.During gastrulation its expression becomes axial and is strongest in theneural tube.

FIG. 4B. qPCR analysis shows that, relative to actin, expression of elais exclusively zygotic, peaking at 100% epiboly and absent by 4 dayspost-fertilization.

FIG. 4C. In zebrafish, ela consists of three exons located onchromosome 1. A custom pair of Zinc Finger Nucleases (ZFNs) targetingexon 1 of ela was used to create an allelic series of mutations withinthe signal peptide of Ela. FIG. 4C discloses SEQ ID NO: 156.

FIG. 4D. Three distinct loss-of-function alleles were generated, theela^(br21)allele results in a unique 7 amino-acid in frame deletion inEla's signal peptide. Alleles ela^(br13) and ela^(br15) caused prematurestop codons and disrupt the reading frame resulting in no Ela maturepeptide. FIG. 4D discloses SEQ ID NOs: 36 and 157-159, respectively, inorder of appearance.

FIG. 4E. Relative to wildtype embryos, homozygous null ela^(br13)embryos show defective epiboly movements and a constricted germ ring atthe involuting margin at 70% epiboly.

FIG. 4F. At 100% epiboly, by RT-PCR endogenous ela and ela^(br13) mRNAare of distinct size. By western blotting using the α C antibody,endogenous Ela is recognized in wt embryos and is absent in nullela^(br13) siblings.

FIGS. 5A-5F. ela Knockout Zebrafish Have Severe Cardiovascular Defects

FIG. 5A. Null ela^(br21) larvae display pericardial oedema (whitearrowheads), accumulated erythrocytes (red arrowheads), and have noblood circulation. Variable posterior anomalies are observed includingloss of ventral fin (black arrowheads), tailbud duplications (inset) andextreme tail/trunk truncations (bottom embryo).

FIG. 5B. Loss of ela causes severe cardiac anomalies ranging from mildheart dysplasia to total heart agenesis as shown by H&E staining onsections from top and bottom embryos shown in A.

FIG. 5C. Null ela^(br13) fish have severe reduction of cmlc1 expressionwhich marks the developing heart.

FIG. 5D. Null ela^(br13) fish display increased hematopoiesis as judgedby the up-regulation of scl, a marker of blood precursors.

FIG. 5E. Classification of null ela^(br13) larvae with varying degreesof tail defects. Class I are defined as having pericardial oedema andtail blood clot, class 2 have ventral fin defects, or tailbudduplications, in addition to class 1 phenotypes and class 3 larvae haveall phenotypes of class 1 combined with mild to severe tail/trunktruncations.

FIG. 5F. Percentages of ela mutant fertile adults that were obtainedfrom heterozygous intercrosses using all three alleles.

FIGS. 6A-6F. ELA is Essential for Endoderm Differentiation

FIG. 6A. Relative to wt embryos, homozygous null ela^(br13) embryos showconvergence-extension defects resulting in delayed blastopore closureand thickened notochord (insets) as indicated by altered bra expression.

FIG. 6B. The loss of ela causes defective migration of mediolateralgata5 expressing cells, which mark mesendoderm cells that guide heartprogenitors to the anterior lateral plate mesoderm.

FIG. 6C. ela mutant embryos show a more compact sox17 expression patternthan do wildtype embryos, sox17+ forerunners cells are not affected.

FIG. 6D. ela null embryos have approximately 40% less sox17+ cells thantheir heterozygous siblings at 75% epiboly.

FIG. 6E. Directed endoderm differentiation of shELA, but not control,hESCs is markedly impaired as judged by the reduction in SOX17expression by immunofluorescence. Addition of recombinant ELA issufficient to rescue this loss of endoderm differentiation potential inshELA hESCs.

FIG. 6F. Quantification of endodermal differentiation efficiency incontrol, shELA and shELA with ELA peptide hESCS after 5 days reveals asignificant 45% reduction of SOX17 expression upon ELA depletion whichcan be entirely rescued by addition of exogenous ELA.

FIGS. 7A-7H. ELA's Cognate Receptor for Endoderm Differentiation IsAPLNR.

FIG. 7A. ELA and APELIN are very basic hormones with isoelectric pointsexceeding 12. FIG. 7A discloses SEQ ID NOs: 160-161, respectively, inorder of appearance.

FIG. 7B. By Q-PCR, the onset of transcription of aplnra and aplnrbcoincides with that of ela at the midblastula transition, apelinexpression debuts 5 hours later during gastrulation.

FIG. 7C. Relative to control embryos, the expression of aplnra andaplnrb at 70% epiboly becomes stronger and confined to the mostequatorial hypoblast in ela mutant (white arrowheads: specific aplnraexpression in the animal pole).

FIG. 7D. Wildtype embryo with a beating heart and blood circulation at 6days post fertilization, normal sox17 expression in definitive endodermat 75% epiboly, no erythrocyte accumulation in the intermediate cellmass, and cmlc1 expression in the heart forming region at 30 hpf.

FIG. 7E. Aplnr morphants phenocopy ela mutant embryos, with no beatingheart, loss of blood circulation at 6 days post fertilization, reducedsox17 expression in definitive endoderm at 75% epiboly, accumulationerythrocytes in the intermediate cell mass and loss of cmlc1 expressionin the heart forming region at 30 hpf.

FIG. 7F. ela mutant embryos are indistinguishable from Aplnr morphantsand have no beating heart nor blood circulation at 6 days postfertilization, show reduced sox17 expression in definitive endoderm at75% epiboly, have accumulated erythrocytes in the intermediate cellmass, and no cmlc1 expression in the heart forming region at 30 hpf.

FIG. 7G. In 293T cells, overexpression of zebrafish aplnra or aplnrb orhuman APLNR is sufficient to confer cell surface binding to recombinantELA conjugated to Alkaline-Phosphatase (AP-ELA).

FIG. 7H. In 293T cells, overexpression of zebrafish aplnrb, but not itsmutant form grinch carrying the W90L missense mutation, or GPR15 anorphan GPCR closely related to APLNR, is enough to afford binding torecombinant ELA conjugated to Alkaline-Phosphatase (AP-ELA).

FIG. 8. Graphical abstract.

FIGS. 9A-9H. Expression of ELA is Highest in Human Blastocysts and hESCsand is Under POU5F1 Control, related to FIG. 1.

FIG. 9A. According to Unigene, ELA is most highly expressed inpre-implantation human blastocysts.

FIG. 9B. According to GEO, ELA expression in the human pre-implantationembryo starts by the blastocyst stage consistent with a zygoticexpression.

FIG. 9C. ELA expression in hESCs is downregulated following POU5F1knockdown.

FIG. 9D. The promoter region of ELA (chr4:165,796,806-165,798,359)transfected in hESCs can only drive expression of a firefly luciferasereporter in the presence of its upstream POU5F1 enhancer (chr4:165,787,570-165,788,797).

FIG. 9E. ELA expression is rapidly downregulated in hESCs upon embryoidbody differentiation.

FIG. 9F. ELA expression is rapidly downregulated in hESCs undergoingdefinitive endoderm differentiation.

FIG. 9G. ELA expression is rapidly downregulated in hESCs undergoingRA-mediated neuroectoderm differentiation.

FIG. 9H. ELA expression is initially downregulated in hESCs undergoingcardiac differentiation, but is re-expressed after day 5.

FIGS. 10A-10H. ELA is Localized to the Golgi in hESCs and its knockdownCompromises Pluripotency, related to FIG. 2.

FIG. 10A. In hESCs, ELA partly co-localizes with TGN46 a maker of thetrans-Golgi network.

FIG. 10B. The same Golgi staining pattern is observed using α Nantibodies.

FIG. 10C. Endogenous ELA in hECs can be efficiently knocked down bysiRNA-mediated depletion.

FIG. 10D. Endogenous ELA in hESCs can be efficiently knocked-down byinducible shRNA-mediated depletion. shRNA against β2M is used as acontrol against non-specific effects of doxycycline treatment or hairpinRNA expression.

FIG. 10E. When 10 million control or shELA hESCs were injectedsubcutaneously into SCID mice (n=6), only control, but not shEL hESCsgave rise to tumors by day 60.

FIG. 10F. shELA hESCs lose expression of pluripotency markers POU51 andNANOG upon serial passaging.

FIG. 10G. Single colony qPCR analysis of passage 4 shELA hESCs confirmsdownregulation of POU51 and NANOG.

FIG. 10H. shRNA-mediated depletion of ELA or β2M does not affect thenumber of cells going through S phase and incorporating EDU.

FIGS. 11A-11B. In hESCs, Recombinant ELA is Bioactive and endogenous ELAcan be Neutralized by the α N Antibody, related to FIGS. 3A-3K.

FIG. 11A. Dose-dependent response of hESCs to recombinant ELA peptide.

FIG. 11B. Addition of purified α N antibodies to hESCs medium inhibitsendogenous ELA and causes similar effects as shELA knockdown. Theneutralizing activity of α N antibodies can be competed out by additionof the mutant non-signaling ELA^(R>GG) peptide.

FIGS. 12A-12D. Homozygous ela^(br21) and ela^(br15) mutant fish haveIdentical Phenotypes, related to FIG. 5.

FIG. 12A. Wildtype larvae at 6 days post-fertilization.

FIG. 12B. The 7 amino-acid in frame mutant ela^(br21) allele causesidentical phenotypes as the two frameshift mutants ela^(br13) andela^(br15).

FIG. 12C. The ela^(br15)mutant fish are indistinguishable from theela^(br21) and ela^(br15) alleles.

FIG. 12D. Overexpression of 200 pg of zebrafish ela ORF mRNA causesheart dysgenesis (open arrows) and accumulation of erythrocytes in theICM (red arrows), similar to ela null fish.

FIGS. 13A-13C. Endoderm Differentiation is Defective in ela mutant fishand ELA-depleted hESCs, related to FIG. 6.

FIG. 13A. ela^(br21) and ela^(br15) mutant fish show similar defects insox17 expression pattern at 75% epiboly.

FIG. 13B. Directed endoderm differentiation of shELA, but not shβ2M andcontrol hESCs, is markedly impaired as judged by the reduction in SOX17expression.

FIG. 13C. Quantification of endodermal differentiation efficiency incontrol, shELA and shβ2M hESCS after directed differentiation reveals asignificant 35% reduction of SOX17 expression upon ELA depletion.

FIGS. 14A-14F. APLNR is Necessary for ELA Binding but is Not the ELAReceptor in hESCs, related to FIG. 7.

FIG. 14A. H3K4me3 by Chip-Seq, transcript levels by RNA seq. and DNAmethylation levels by MeDIP around the ELA and APLNR loci in hESCs. Dataare extracted from the Human Epigenome Atlas (www.genboree.org).

FIG. 14B. qPCR analysis of APLNR transcript levels (normalized to GAPDH)in Day 0 undifferentiated hESCs compared to Day 3 differentiatedmesoendoderm cells. APLNR transcript levels are significantly reduced byshRNA-mediated knockdown using two different constructs.

FIG. 14C. Day 0 undifferentiated hESCs are uniformly APLNR-negative,whereas levels of APLNR in Day 3 mesoendoderm cells are one log higher,as measured by FACs. Grey shaded histograms are cells stained only withthe secondary antibody. These results are confirmed using a second lineof hESCs HES3.

FIG. 14D. shAPLNR Day 3 mesoendoderm cells have significantly reducedcell surface APLNR levels. No changes are observed in Day 0undifferentiated hESCs, confirming the absence of APLNR.

FIG. 14E. shAPLNR Day 3 mesoendoderm cells have significantly reducedAP-ELA binding. This reduction is proportional to the decrease in levelsof cell surface APLNR. shAPLNR does not affect the levels of AP-ELAbound to Day 0 undifferentiated hESCs.

FIG. 14F. Undifferentiated shAPLNR hESCs do not show impaired growthcompared to shControl hESCs, as measured on the xCELLigence platform.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA and immunology, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature. See, for example, J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al. (1995 and periodic supplements; Current Protocols inMolecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation andSequencing: Essential Techniques, John Wiley & Sons; J. M. Polak andJames O′D. McGee, 1990, In Situ Hybridization: Principles and Practice;Oxford University Press; M. J. Gait (Editor), 1984, OligonucleotideSynthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E.Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesisand Physical Analysis of DNA Methods in Enzymology, Academic Press;Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by EdwardHarlow, David Lane, Ed Harlow (1999, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow(Editor), David Lane (Editor) (1988, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-314-2), 1855. Handbook of Drug Screening, edited byRamakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, N.Y.,Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes,Reagents, and Other Reference Tools for Use at the Bench, Edited JaneRoskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN0-87969-630-3. Each of these general texts is herein incorporated byreference.

SEQUENCE LISTINGS

SEQ ID NO: 1 shows a sequence of a ELABELA polypeptide signaturesequence. SEQ ID NO: 2 shows a sequence of a Homo ELABELA maturepolypeptide. SEQ ID NO: 3 shows a sequence of a Peromyscus ELABELAmature polypeptide. SEQ ID NO: 4 shows a sequence of a Rattus ELABELAmature polypeptide.

SEQ ID NO: 5 shows a sequence of a Mus ELABELA mature polypeptide. SEQID NO: 6 shows a sequence of a Bos ELABELA mature polypeptide. SEQ IDNO: 7 shows a sequence of a Sus ELABELA mature polypeptide. SEQ ID NO: 8shows a sequence of a Dasypus ELABELA mature polypeptide. SEQ ID NO: 9shows a sequence of a Trichosurus ELABELA mature polypeptide.

SEQ ID NO: 10 shows a sequence of a Gallus ELABELA mature polypeptide.SEQ ID NO: 11 shows a sequence of a Gekko ELABELA mature polypeptide.SEQ ID NO: 12 shows a sequence of a Anolis ELABELA mature polypeptide.SEQ ID NO: 13 shows a sequence of a Xenopus ELABELA mature polypeptide.SEQ ID NO: 14 shows a sequence of a Ambystoma ELABELA maturepolypeptide.

SEQ ID NO: 15 shows a sequence of a Oryzias ELABELA mature polypeptide.SEQ ID NO: 16 shows a sequence of a Callorhinchus ELABELA maturepolypeptide. SEQ ID NO: 17 shows a sequence of a Oncorhynchus ELABELAmature polypeptide. SEQ ID NO: 18 shows a sequence of a Danio ELABELAmature polypeptide.

SEQ ID NO: 19 shows a sequence of a Human ELABELA signal sequence. SEQID NO: 20 shows a sequence of a Homo ELABELA polypeptide with signalsequence (bold). SEQ ID NO: 21 shows a sequence of a Peromyscus ELABELApolypeptide with signal sequence (bold). SEQ ID NO: 22 shows a sequenceof a Rattus ELABELA polypeptide with signal sequence (bold).

SEQ ID NO: 23 shows a sequence of a Mus ELABELA polypeptide with signalsequence (bold). SEQ ID NO: 24 shows a sequence of a Bos ELABELApolypeptide with signal sequence (bold). SEQ ID NO: 25 shows a sequenceof a Sus ELABELA polypeptide with signal sequence (bold). SEQ ID NO: 26shows a sequence of a Dasypus ELABELA polypeptide with signal sequence(bold). SEQ ID NO: 27 shows a sequence of a Trichosurus ELABELApolypeptide with signal sequence (bold).

SEQ ID NO: 28 shows a sequence of a Gallus ELABELA polypeptide withsignal sequence (bold). SEQ ID NO: 29 shows a sequence of a GekkoELABELA polypeptide with signal sequence (bold). SEQ ID NO: 30 shows asequence of a Anolis ELABELA polypeptide with signal sequence (bold).SEQ ID NO: 31 shows a sequence of a Xenopus ELABELA polypeptide withsignal sequence (bold). SEQ ID NO: 32 shows a sequence of a AmbystomaELABELA polypeptide with signal sequence (bold).

SEQ ID NO: 33 shows a sequence of a Oryzias ELABELA polypeptide withsignal sequence (bold). SEQ ID NO: 34 shows a sequence of aCallorhinchus ELABELA polypeptide with signal sequence (bold). SEQ IDNO: 35 shows a sequence of a Oncorhynchus ELABELA polypeptide withsignal sequence (bold). SEQ ID NO: 36 shows a sequence of a DanioELABELA polypeptide with signal sequence (bold).

SEQ ID NO: 37 shows a Human (Homo sapiens) ELABELA cDNA sequence. SEQ IDNO: 38 shows a Mouse (Mus musculus) ELABELA cDNA sequence. SEQ ID NO: 39shows a Chicken (Gallus gallus) ELABELA cDNA sequence. SEQ ID NO: 40shows a Xenopus (Xenopus laevis) ELABELA cDNA sequence. SEQ ID NO: 41shows a Zebrafish (Danio rerio) ELABELA cDNA sequence.

SEQ ID NO: 42 shows a Human (Homo sapiens) ELABELA genomic sequence. SEQID NO: 43 shows a Mouse (Mus musculus) ELABELA genomic sequence. SEQ IDNO: 44 shows a Chicken (Gallus gallus) ELABELA genomic sequence. SEQ IDNO: 45 shows a Xenopus (Xenopus laevis) ELABELA genomic sequence. SEQ IDNO: 46 shows a Zebrafish (Danio rerio) ELABELA genomic sequence.

SEQ ID NO: 47 shows a Anti-ELABELA shRNA sequence A. SEQ ID NO: 48 showsa Anti-ELABELA shRNA sequence B. SEQ ID NO: 49 shows a Anti-ELABELAshRNA sequence C. SEQ ID NO: 50 shows a Anti-ELABELA shRNA sequence D.SEQ ID NO: 51 shows a Anti-ELABELA shRNA sequence E.

DETAILED DESCRIPTION

In describing the different ELABELA polypeptides variants encompassed bythis document, the following nomenclature will be adopted for ease ofreference:

-   -   (i) where the substitution includes a number and a letter, e.g.,        31A, then this refers to [position according to the numbering        system/substituted amino acid]. Accordingly, for example, the        substitution of an amino acid to alanine in position 31 is        designated as 31A;    -   (ii) where the substitution includes a letter, a number and a        letter, e.g., R31A, then this refers to [original amino        acid/position according to the numbering system/substituted        amino acid]. Accordingly, for example, the substitution of        arginine with alanine in position 31 is designated as R31A.

Where two or more possible substituents are possible at a particularposition, this will be designated by contiguous letters, which canoptionally be separated by slash marks “/”, e.g., R31A/G or K32A/G.Where the relevant amino acid at a position can be substituted by anyamino acid, this is designated by [position according to the numberingsystem/X], e.g., 31X.

Multiple mutations can be designated by being separated by slash marks“/”, e.g. R31A/K32G representing mutations in position 31 and 32substituting arginine with alanine and lysine with glycine respectively.

ELABELA

We disclose a previously unknown 54-amino acid hormone with a predictedsignal peptide. The peptide hormone, together with its variants,homologues, derivatives and fragments will generally be referred to inthis document as ELABELA (abbreviated to ELA). This peptide hormone ispresent in all vertebrate species.

In vivo, ELA expression is highest in the human blastocyst (hs.105196,LOC100506013), and has been reported to be rapidly down-regulated duringhESC differentiation (Miura et al., 2004).

To our knowledge, the next earliest known peptide hormone to beexpressed during embryogenesis is APELIN (APLN), the transcription ofwhich begins during gastrulation in mice (D'Aniello et al., 2009).However, Apln knockout mice have no defects in early embryonicdevelopment (Kuba et al., 2007), which is inconsistent with theinactivation of its receptor Aplnr (also known as Apj or Agtrl1) whoseloss leads to variable embryonic lethality due to growth retardation andcardiac malformations (Charo et al., 2009).

In zebrafish, mutations in the APLNR homologue aplnrb impair themigration of cardiac progenitors from the lateral plate mesoderm intothe heart field. Because aplnra and aplnrb are expressed in earlyprecursors of the endodermal lineage, hours before the onset of aplnexpression, it has been postulated that APLNR transduces the signal ofan earlier hormone, yet to be discovered (Charo et al., 2009; Scott etal., 2007).

Here, we demonstrate that ELA is a novel secreted peptide hormone. Usingzinc-finger-nuclease (ZFN)-mediated gene inactivation, we created anallelic series of ela null zebrafish, and show that it is essential forendoderm differentiation and heart morphogenesis.

ELABELA, its variants, homologues, derivatives and fragments, as well asmodulators such as agonists and antagonists, can therefore be used forthe treatment, prophylaxis or alleviation of an ELABELA associatedcondition. ELABELA associated conditions are described in further detailelsewhere in this document and can comprise a cardiac dysfunction orcardiovascular disease or a condition associated with high bloodpressure, for example.

We find that Ela, and not Apln, is the first ligand recognized by Aplnrwhich mediates its effect for endoderm differentiation and subsequentcardiogenesis. However, in hESCs where APLNR is not expressed, we showthat ELA serves as an endogenous signal that protects against apoptosisand maintains the self-renewal capacity of hESCs.

Together, our results unveil the existence of a hitherto uncharacterizedhormone ELA that is indispensable for the self-renewal of cultured hESCsand critical in vivo for heart morphogenesis via the APLNR signalingpathway.

ELABELA Polypeptides

The methods and compositions described here make use of ELABELApolypeptides.

We demonstrate that ELABELA is expressed in a number of vertebratespecies. We further demonstrate that ELABELA is highly conserved betweenspecies, and that the ELABELA polypeptide sequence comprises a number ofconserved regions.

The sequence alignment below shows ELABELA proteins across 17 vertebratespecies. Conserved residues are shown in bold and are alsohighlighted/shaded. Signal sequence is shown in italics. The sequencesin Table D1 correspond to those in SEQ ID NO: 20 to 34 of the sequencelistings. (“Invariant residues” consensus sequence disclosed as SEQ IDNO: 56).

Positions r/ Homo1-----------------------------3131------39---4344---48495051525354Invariant residues

Homo

Peromyscus

Rattus

Mus

Bos

Sus

Dasypus

Trichosurus

Gallus

Gekko

Anolis

Xenopus

Ambystoma

Oryzias

Callorhinchus

A number of sequences from other species show small variations inlength:

For example, the Oncorhynchus ELABELA sequence (SEQ ID NO: 35) has a oneamino-acid deletion between underlined TV residues. The missing orcorresponding residue is Methionine 15 in the human sequence. This iswithin the signal peptide.

As another example, the Danio rerio ELABELA sequence (SEQ ID NO: 36) hasa four amino-acid insertion underlined as DKHG (SEQ ID NO: 57). These 4residues are predicted to be kept in the mature peptide.

In the broadest sense, an ELABELA polypeptide is a polypeptide thatincludes an “ELABELA signature” sequence. In preferred embodiments ofthe aspects described herein, an ELABELA polypeptide can furthercomprise one or more activities, such as a biological activity of anative ELABELA polypeptide, as described herein. As is clear from thesequence alignment, a number of ELABELA signatures are possible.

For example, an ELABELA signature can comprise the sequence HSRVPFP (SEQID NO: 58). Accordingly, as used in this document, the term “ELABELApolypeptide” can mean a polypeptide which comprises an HSRVPFP sequence(SEQ ID NO: 58). In preferred embodiments of the aspects describedherein, an ELABELA polypeptide comprising SEQ ID NO: 58 comprises one ormore biological activities of a native ELABELA polypeptide, such asmaintaining self-renewal, pluripotency, or both of a stem cell, asdescribed herein.

Alternatively, or in addition, an ELABELA signature can comprise thesequence RCXXXHSRVPFP (SEQ ID NO: 59). In this sense, therefore term“ELABELA polypeptide” can mean a polypeptide which comprises anRCXXXHSRVPFP (SEQ ID NO: 59) sequence, in which X represents any aminoacid residue. In preferred embodiments of the aspects described herein,an ELABELA polypeptide comprising SEQ ID NO: 59 comprises one or morebiological activities of a native ELABELA polypeptide, such asmaintaining self-renewal, pluripotency, or both of a stem cell, asdescribed herein.

In preferred embodiments, however, the ELABELA signature is intended torefer to a sequence CXXXRCXXXHSRVPFP (SEQ ID NO: 1), in which Xsignifies an amino acid residue. Accordingly, the term “ELABELApolypeptide” as used in this document is intended to refer to a sequencecomprising a CXXXRCXXXHSRVPFP (SEQ ID NO: 1), in which X signifies anamino acid residue. In preferred embodiments of the aspects describedherein, an ELABELA polypeptide comprising SEQ ID NO: 1 comprises one ormore biological activities of a native ELABELA polypeptide, such asmaintaining self-renewal, pluripotency, or both of a stem cell, asdescribed herein.

For the purposes of this document, the term “ELABELA polypeptide” shouldalso be taken to encompass any fragment, homologue, variant orderivative of such a polypeptide. Such ELABELA fragments, homologues,variants and derivatives are described in further detail elsewhere inthis document. In preferred embodiments of the aspects described herein,such ELABELA fragments, homologues, variants and derivatives compriseone or more biological activities of a native ELABELA polypeptide, suchas maintaining self-renewal, pluripotency, or both of a stem cell, asdescribed herein.

The ELABELA polypeptide encompassed by this document can thereforecomprise an signature or conserved region from any of the vertebratespecies in which ELABELA is expressed (see for example FIG. 1C and thesequence alignment above). Such signatures and conserved regions are setout as SEQ ID NO: 2 to SEQ ID NO: 18 and SEQ ID NOs: 60-76.

The ELABELA polypeptide can comprise a signature or conserved regionfrom any species, for example: a Homo sequence CLQRRCMPLHSRVPFP (SEQ IDNO: 60); a Peromyscus sequence CFRRRCVPLHSRVPFP (SEQ ID NO: 61); aRattus sequence CFRRRCISLHSRVPFP (SEQ ID NO: 62); a Mus sequenceCFRRRCIPLHSRVPFP (SEQ ID NO: 63); a Bos sequence CLQRRCMPLHSRVPFP (SEQID NO: 64); a Sus sequence CLQRRCMPLHSRVPFP (SEQ ID NO: 65); a Dasypussequence CFQRRCMPLHSRVPFP (SEQ ID NO: 66); a Trichosurus sequenceCPQRRCMPLHSRVPFP (SEQ ID NO: 67); a Gallus ELABELA polypeptide sequenceCSHRRCMPLHSRVPFP (SEQ ID NO: 68); a Gekko sequence CSHRRCMPLHSRVPFP (SEQID NO: 69); a Anolis sequence CSHRRCMPLHSRVPFP (SEQ ID NO: 70); aXenopus sequence CFLKRCIPLHSRVPFP (SEQ ID NO: 71); a Ambystoma sequenceCSLRRCMPLHSRVPFP (SEQ ID NO: 72); a Oryzias sequence CLHRRCMPLHSRVPFP(SEQ ID NO: 73); a Callorhinchus sequence CWHRRCLPFHSRVPFP (SEQ ID NO:74); a Oncorhynchus sequence CPHRRCMPLHSRVPFP (SEQ ID NO: 75); a Daniosequence CPKKRCLPLHSRVPFP (SEQ ID NO: 76).

The ELABELA polypeptide can therefore comprise a signature from humanELABELA, i.e., CXXXRCXXXHSRVPFP (SEQ ID NO: 1) can compriseCLQRRCMPLHSRVPFP (SEQ ID NO: 60). It can comprise a signature from mouseELABELA, i.e., CXXXRCXXXHSRVPFP (SEQ ID NO: 1) can compriseCFRRRCIPLHSRVPFP (SEQ ID NO: 63). In preferred embodiments of theaspects described herein, an ELABELA polypeptide comprising SEQ ID NO:60 or SEQ ID NO: 63 comprises one or more biological activities of anative ELABELA polypeptide, such as maintaining self-renewal,pluripotency, or both of a stem cell, as described herein.

A number of other residues can also be present in the ELABELApolypeptide. For example, the ELABELA polypeptide can comprise one ormore basic residues upstream of the ELABELA signature sequence.

In particular, the ELABELA polypeptide can comprise a basic residue ator about position −7 upstream of the ELABELA signature sequenceCXXXRCXXXHSRVPFP (SEQ ID NO: 1). The basic residue can comprise a lysineresidue, or an arginine residue.

Accordingly, the ELABELA polypeptide can comprise a sequence (R/K)CXXXRCXXXHSRVPFP (SEQ ID NO: 77) where X represents any amino acid. Inpreferred embodiments of the aspects described herein, an ELABELApolypeptide comprising SEQ ID NO: 77 comprises one or more biologicalactivities of a native ELABELA polypeptide, such as maintainingself-renewal, pluripotency, or both of a stem cell, as described herein.

The ELABELA polypeptide can, alternatively or in addition, comprise abasic residue at or about position −8 upstream of the ELABELA signaturesequence CXXXRCXXXHSRVPFP (SEQ ID NO: 1). The basic residue can comprisea lysine residue, or an arginine residue.

Accordingly, the ELABELA polypeptide can comprise a sequence (R/K)CXXXRCXXXHSRVPFP (SEQ ID NO: 78), where X represents any amino acid. Inpreferred embodiments of the aspects described herein, an ELABELApolypeptide comprising SEQ ID NO: 78 comprises one or more biologicalactivities of a native ELABELA polypeptide, such as maintainingself-renewal, pluripotency, or both of a stem cell, as described herein.

As noted above, the ELABELA polypeptide can comprise a pair of basicresidues at or about positions −7 and −8 upstream of the ELABELAsignature sequence CXXXRCXXXHSRVPFP (SEQ ID NO: 1), i.e., it cancomprise a sequence (R/K)(R/K) CXXXRCXXXHSRVPFP (SEQ ID NO: 79).

It will be evident that where the ELABELA polypeptide comprises a signalsequence (see below), position −8 upstream of the ELABELA signaturesequence CXXXRCXXXHSRVPFP (SEQ ID NO: 1) corresponds to position 31 of ahuman ELABELA sequence (SEQ ID NO: 20) and position −7 upstream of theELABELA signature sequence CXXXRCXXXHSRVPFP (SEQ ID NO: 1) correspondsto position 32 of a human ELABELA sequence (SEQ ID NO: 20).

The ELABELA polypeptide can comprise a sequence selected from the groupconsisting of: SEQ ID NO: 2 to SEQ ID NO: 18. The ELABELA polypeptidecan comprise a human ELABELA sequence shown as SEQ ID NO: 2. It cancomprise a mouse ELABELA sequence shown as SEQ ID NO: 5.

In some embodiments, the ELABELA polypeptide comprises a signal peptideor signal sequence. The skilled reader will appreciate that the presenceof a signal peptide will allow the ELABELA peptide to be exported andsecreted from a cell. The skilled reader will also know how to engineersuch signal sequences into the sequences of ELABELA polypeptidesdescribed in this document.

ELABELA polypeptides comprising signal sequences can be referred to inthis document for convenience as “full length” polypeptides. They may beproduced by including any known signal sequences, including the ELABELAsignal sequences disclosed in this document in an ELABELA polypeptide tobe produced.

The ELABELA polypeptide can comprise an ELABELA signal sequence from anysuitable species, for example, Homo MRFQQFLFAFFIFIMSLLLISG (SEQ ID NO:19); Peromyscus MRFQHYFLVFFIFAMSLLFITE (SEQ ID NO: 80); RattusMRFQPLFWVFFIFAMSLLFITE (SEQ ID NO: 81); Mus MRFQPLFWVFFIFAMSLLFISE (SEQID NO: 82); Bos MRFHQFFLLFVIFMLSLLLIHG (SEQ ID NO: 83); SusMRFRQFFLVFFIFMMNLLLICG (SEQ ID NO: 84); Dasypus MKFQQFFYVFFVFIMSLLLING(SEQ ID NO: 85); Trichosurus MRFQLLFFLFLFFTMGILLIDG (SEQ ID NO: 86);Gallus MRLRRLLCVVFLLLVSLLPAAA (SEQ ID NO: 87); GekkoMRLQLLLLTCFLILTGVLLGNG (SEQ ID NO: 88); Anolis MRLQQLLLTWFLLLAGALLING(SEQ ID NO: 89); Xenopus MDFQKLLYALFFILMSLLLING (SEQ ID NO: 90);Ambystoma MKWQKLLAILFWILMGALLVNG (SEQ ID NO: 91); OryziasMRVWNLLYLLLLLAAALAPVFS (SEQ ID NO: 92); or CallorhinchusMRFQHLLHIILLLCTSLLLISG (SEQ ID NO: 93).

It can for example comprise a human ELABELA signal sequence shown as SEQID NO: 19, i.e., MRFQQFLFAFFIFIMSLLLISG.

Examples of “full length” or “native” ELABELA polypeptides are disclosedherein, and include any of the sequences set out as SEQ ID NO: 20 to SEQID NO: 36. In some embodiments, the ELABELA polypeptide can comprise orconsist of a human ELABELA polypeptide having or comprising a sequenceshown as SEQ ID NO: 20. It can comprise or consist of a mouse ELABELApolypeptide, such as the sequence having SEQ ID NO: 23.

An “ELABELA polypeptide” described herein can preferably comprise one ormore activities of a native ELABELA polypeptide, such as one or morebiological activities of a native ELABELA polypeptide. Such ELABELAactivities are described in detail elsewhere in this document, andinclude, for example, the ability to maintain self-renewal orpluripotency, or both, of a cell such as a stem cell.

As noted above, homologues variants and derivatives thereof of any, someor all of these polypeptides are also included in the term “ELABELApolypeptide”.

For example, an ELABELA polypeptide can comprise one or more reducedcysteines having a sulfhydryl group. The reduced cysteines can appearanywhere in the ELABELA amino acid sequence. For example, a cysteine atposition 1 with reference to the numbering in the sequenceCXXXRCXXXHSRVPFP (SEQ ID NO: 1) can comprise a reduced cysteine having asulfhydryl group. A cysteine at position 6 can similarly comprise areduced cysteine shaving a sulfhydryl group. The cysteine residues atboth position 1 and position 6 can be so modified. The ELABELApolypeptide can comprise an intramolecular covalent bond between thecysteine residues at positions 1 and 6, with reference to the numberingin the sequence CXXXRCXXXHSRVPFP (SEQ ID NO: 1).

The position numberings above correspond respectively to positionnumbers 39 and 43 respectively in the human ELABELA polypeptide sequenceMRFQQFLFAFFIFIMSLLLISGQRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP (SEQ ID NO: 20).

As another example, an ELABELA polypeptide can comprise one or moremutations of any of the sequences discussed in this document, such asthose referred to as “ELABELA polypeptides”. Such mutated sequences aredescribed in further detail elsewhere in this document.

Included are ELABELA polypeptides which comprise a mutation of a basicresidue at position 31 of their sequences, with reference to theposition numbering of a human ELABELA sequence shown as SEQ ID NO: 20.The basic residue at position 31 can be mutated to a neutral residue.For example, an arginine or lysine residue at position 31 can be mutatedto an alanine or glycine residue.

The ELABELA polypeptide can comprise a mutation of a basic residue atposition 32, with reference to the position numbering of a human ELABELAsequence shown as SEQ ID NO: 20. For example, the basic residue atposition 32 can be mutated to a neutral residue. Thus, an arginine orlysine residue at position 32 can be mutated to an alanine or glycineresidue.

The ELABELA polypeptide can comprise a mutant in which both of the basicresidues set out above are so mutated. Thus, the ELABELA polypeptide cancomprise an R31G, R31A, K31G or K31A substitution. The substitutions cancomprise R32G, R32A, K32G or K32A. The ELABELA polypeptide can thereforecomprise any one of the sequences: (R/K)(A/G)XXXXXXCXXXRCXXXHSRVPFP (SEQID NO: 94), (A/G)(R/K) XCXXXRCXXXHSRVPFP (SEQ ID NO: 95) or (A/G)(A/G)CXXXRCXXXHSRVPFP (SEQ ID NO: 96).

As noted above, the position numbering is with reference to the positionnumbering of a human ELABELA sequence shown as SEQ ID NO: 20.

It will be appreciated that, with reference to the position numbering ofa human ELABELA sequence shown as SEQ ID NO: 20, positions 31 and 32correspond to positions −8 and −7 respectively with respect to theposition numbering of the ELABELA signature sequence CXXXRCXXXHSRVPFP(SEQ ID NO: 1).

As each of the ELABELA polypeptide sequences described in this documentnecessarily comprise an ELABELA signature sequence, the skilled personwill be able to establish the position numbering of any particularresidue within ELABELA polypeptide sequence in his possession. That isto say, a skilled person will, given the information available in thisdocument, and in other resources he has in his possession, be able toestablish, in any ELABELA polypeptide sequence, the position numberingof any particular residue with reference to the human ELABELA sequenceshown as SEQ ID NO: 20 or with reference to the position numbering ofthe ELABELA signature sequence CXXXRCXXXHSRVPFP (SEQ ID NO: 1) comprisedin the ELABELA polypeptide.

ELABELA polypeptides can be used for a variety of means, for example,administration to an individual suffering from, or suspected to besuffering from an ELABELA associated condition, for the treatmentthereof.

They can also be used for production or screening of anti-ELABELA agentssuch as specific ELABELA binding agents, in particular, anti-ELABELAantibodies. These are described in further detail elsewhere in thisdocument.

The expression of ELABELA polypeptides can be detected for diagnosis ordetection of an ELABELA associated condition.

ELABELA associated conditions are described in further detail elsewherein this document.

Polypeptide

A “polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidescan contain amino acids other than the 20 gene-encoded amino acids.

“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification can be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide can contain many types of modifications.

ELABELA polypeptides can be branched as a result of ubiquitination, andthey can be cyclic, with or without branching. Cyclic, branched andbranched cyclic polypeptides can result from posttranslation naturalprocesses or can be made by synthetic methods. Modifications includeacetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-inking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-inks, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, Proteins—Structure and MolecularProperties, 2nd Ed., T. E. Creighton, W.H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., “Analysis for protein modifications and nonproteincofactors”, Meth Enzymol (1990) 182:626-646 and Rattan et aL, “ProteinSynthesis: Posttranslational Modifications and Aging”, Ann NY Acad Sci(1992) 663:48-62.

In some embodiments of the aspects described herein, an ELABELApolypeptide comprises a fragment having the sequence of SEQ ID NO: 53.In some such embodiments, the fragment comprises a pyroglutamate at theN-terminus. As understood by one of ordinary skill in the art, whenglutamatic acid or glutamine are at the N-terminus of a polypeptide,such as an ELABELA polypeptide fragment of SEQ ID NO: 53 describedherein, they can spontaneously cyclize to form pyroglutamate. In someembodiments of the aspects described herein, the fragment having thesequence of SEQ ID NO: 53 further comprises a label.

The term “polypeptide” includes the various synthetic peptide variationsknown in the art, such as a retroinverso D peptides. The peptide can bean antigenic determinant and/or a T-cell epitope. The peptide can beimmunogenic in vivo. The peptide can be capable of inducing neutralisingantibodies in vivo.

As applied to ELABELA, the resultant amino acid sequence can have one ormore activities, such as biological activities in common with a ELABELApolypeptide, for example a human ELABELA polypeptide. ELABELApolypeptide activities are described in detail elsewhere in thisdocument. As an example, a ELABELA homologue can be capable ofmaintaining self-renewal or pluripotency, or both, of a cell such as astem cell.

In particular, the term “homologue” is intended to cover identity withrespect to structure and/or function providing the resultant amino acidsequence has ELABELA activity. With respect to sequence identity (i.e.similarity), there can be at least 70%, such as at least 75%, such as atleast 85%, such as at least 90% sequence identity. There can be at least95%, such as at least 98%, sequence identity. These terms also encompasspolypeptides derived from amino acids which are allelic variations ofthe ELABELA nucleic acid sequence.

Other ELABELA Polypeptides

ELABELA variants, homologues, derivatives and fragments are also of usein the methods and compositions described here.

The terms “variant”, “homologue”, “derivative” or “fragment” in relationto ELABELA include any substitution of, variation of, modification of,replacement of, deletion of or addition of one (or more) amino acid fromor to a sequence. Unless the context admits otherwise, references to“ELABELA” includes references to such variants, homologues, derivativesand fragments of ELABELA. In preferred embodiments of the aspectsdescribed herein, such ELABELA fragments, homologues, variants andderivatives comprise one or more biological activities of a nativeELABELA polypeptide, such as maintaining self-renewal, pluripotency, orboth of a stem cell, as described herein.

As used herein a “deletion” is defined as a change in either nucleotideor amino acid sequence in which one or more nucleotides or amino acidresidues, respectively, are absent.

As used herein an “insertion” or “addition” is that change in anucleotide or amino acid sequence which has resulted in the addition ofone or more nucleotides or amino acid residues, respectively, ascompared to the naturally occurring substance.

As used herein “substitution” results from the replacement of one ormore nucleotides or amino acids by different nucleotides or amino acids,respectively.

ELABELA polypeptides as described here can also have deletions,insertions or substitutions of amino acid residues which produce asilent change and result in a functionally equivalent amino acidsequence.

Deliberate amino acid substitutions can be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine,glycine, alanine, asparagine, glutamine, serine, threonine,phenylalanine, and tyrosine.

Conservative substitutions can be made, for example according to thetable below Amino acids in the same block in the second column and inthe same line in the third column can be substituted for each other:

ALIPHATIC Non-polar G A P I L V Polar—uncharged C S T M N QPolar—charged D E K R AROMATIC H F W Y

ELABELA polypeptides can further comprise heterologous amino acidsequences, typically at the N-terminus or C-terminus, such as theN-terminus.

Heterologous sequences can include sequences that affect intra orextracellular protein targeting (such as leader sequences). Heterologoussequences can also include sequences that increase the immunogenicity ofthe ELABELA polypeptide and/or which facilitate identification,extraction and/or purification of the polypeptides. Another heterologoussequence that can be used is a polyamino acid sequence such aspolyhistidine which can be N-terminal A polyhistidine sequence of atleast 10 amino acids, such as at least 17 amino acids but fewer than 50amino acids can be employed.

The ELABELA polypeptides can be in the form of the “mature” protein orcan be a part of a larger protein such as a fusion protein. It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, pro-sequences, sequences which aid inpurification such as multiple histidine residues, or an additionalsequence for stability during recombinant production.

The signal sequence (secretory sequence or leader sequence) can comprisethe sequence MRFQQFLFAFFIFIMSLLLISG (SEQ ID NO: 19). An example of anELABELA polypeptide which comprises such a signal sequence is the fulllength human ELABELA polypeptide sequence shown as SEQ ID NO: 20.

ELABELA polypeptides as described here are advantageously made byrecombinant means, using known techniques. However they can also be madeby synthetic means using techniques well known to skilled persons suchas using chemical methods, such as solid phase synthesis.

Such polypeptides can also be produced as fusion proteins, for exampleto aid in extraction and purification. Examples of fusion proteinpartners include glutathione-S-transferase (GST), 6×His (SEQ ID NO: 97),GAL4 (DNA binding and/or transcriptional activation domains) andβ-galactosidase.

It can also be convenient to include a proteolytic cleavage site betweenthe fusion protein partner and the protein sequence of interest to allowremoval of fusion protein sequences, such as a thrombin cleavage site.The fusion protein can be one which does not hinder the function of theprotein of interest sequence.

The ELABELA polypeptides can be in a substantially isolated form. Thisterm is intended to refer to alteration by the hand of man from thenatural state. If an “isolated” composition or substance occurs innature, it has been changed or removed from its original environment, orboth. For example, a polynucleotide, nucleic acid or a polypeptidenaturally present in a living animal is not “isolated,” but the samepolynucleotide, nucleic acid or polypeptide separated from thecoexisting materials of its natural state is “isolated”, as the term isemployed herein.

It will however be understood that the ELABELA protein can be mixed withcarriers or diluents which will not interfere with the intended purposeof the protein and still be regarded as substantially isolated. AELABELA polypeptide can also be in a substantially purified form, inwhich case it will generally comprise the protein in a preparation inwhich more than 90%, for example, 95%, 98% or 99% of the protein in thepreparation is a ELABELA polypeptide.

By aligning ELABELA sequences from different species, it is possible todetermine which regions of the amino acid sequence are conserved betweendifferent species (“homologous regions”), and which regions vary betweenthe different species (“heterologous regions”).

An example of such an alignment is set out in FIG. 1C.

The ELABELA polypeptide can comprise a sequence which corresponds to atleast part of a homologous region.

A homologous region shows a high degree of homology between at least twospecies. The two species can comprise for example human and anotherspecies, such as Peromyscus, Rattus, Mus, Bos, Sus, Dasypus,Trichosurus, Gallus, Gekko, Anolis, Xenopus, Ambystoma, Oryzias,Callorhinchus, Oncorhynchus or Danio.

The homologous region can for example show at least 70%, at least 80%,at least 90% or at least 95% identity at the amino acid level using thetests described above.

Examples of homologous regions are set out in this document and cancomprise for example HSRVPFP (SEQ ID NO: 58), RCXXXHSRVPFP (SEQ ID NO:59) or CXXXRCXXXHSRVPFP (SEQ ID NO: 1). In preferred embodiments of theaspects described herein, such homologous regions comprise one or morebiological activities of a native ELABELA polypeptide, such asmaintaining self-renewal, pluripotency, or both of a stem cell, asdescribed herein.

Peptides which comprise a sequence which corresponds to a homologousregion can be used in therapeutic strategies as explained in furtherdetail elsewhere in this document. Alternatively, the ELABELA peptidecan comprise a sequence which corresponds to at least part of aheterologous region. A heterologous region shows a low degree ofhomology between at least two species.

ELABELA Homologues

The ELABELA polypeptides disclosed for use include homologous sequencesobtained from any source, for example related viral/bacterial proteins,cellular homologues and synthetic peptides, as well as variants orderivatives thereof.

Thus polypeptides also include those encoding homologues of ELABELA fromother species including animals such as mammals (e.g. mice, rats orrabbits), especially primates, more especially humans. Morespecifically, homologues include human homologues.

In the context of this document, a homologous sequence is taken toinclude an amino acid sequence which is at least 15, 20, 25, 30, 40, 50,at least 60, at least 70, at least 80 or at least 90% identical, such asat least 95 or at least 98% identical at the amino acid level, forexample over at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59 or 60 or more amino acids with thesequence of a relevant ELABELA sequence.

In particular, homology should typically be considered with respect tothose regions of the sequence known to be essential for protein functionrather than non-essential neighbouring sequences. This is especiallyimportant when considering homologous sequences from distantly relatedorganisms.

Examples of such regions in ELABELA are shown underlined in the sequencebelow:

(SEQ ID NO: 79) (R/K)(R/K)XXXXXXCXXXRCXXXHSRVPFP

Although homology can also be considered in terms of similarity (i.e.amino acid residues having similar chemical properties/functions), inthe context of the present document homology can be expressed in termsof sequence identity.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. These publiclyand commercially available computer programs can calculate % identitybetween two or more sequences.

% identity can be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues (for example less than 50 contiguousamino acids).

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local identity or similarity.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,the default values can be used when using such software for sequencecomparisons. For example when using the GCG Wisconsin Bestfit package(see elsewhere in this document) the default gap penalty for amino acidsequences is −12 for a gap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Altschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). The GCG Bestfit program can be used.

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). The public default values for theGCG package can be used, or in the case of other software, the defaultmatrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, such as % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

The terms “variant” or “derivative” in relation to amino acid sequencesincludes any substitution of, variation of, modification of, replacementof, deletion of or addition of one (or more) amino acids from or to thesequence providing the resultant amino acid sequence retainssubstantially the same activity as the unmodified sequence, such ashaving at least the same activity as the ELABELA polypeptides. Inpreferred embodiments of the aspects described herein, such ELABELAvariants and derivatives comprise one or more biological activities of anative ELABELA polypeptide, such as maintaining self-renewal,pluripotency, or both of a stem cell, as described herein.

Polypeptides having the ELABELA amino acid sequence disclosed here, orfragments or homologues thereof can be modified for use in the methodsand compositions described here. Typically, modifications are made thatmaintain the biological activity of the sequence. Amino acidsubstitutions can be made, for example from 1, 2 or 3 to 10, 20 or 30substitutions provided that the modified sequence retains the biologicalactivity of the unmodified sequence. Alternatively, modifications can bemade to deliberately inactivate one or more functional domains of thepolypeptides described here Amino acid substitutions can include the useof non-naturally occurring analogues, for example to increase bloodplasma half-life of a therapeutically administered polypeptide.

ELABELA Fragments

Polypeptides for use in the methods and compositions described here alsoinclude fragments of the full length sequence of any of the ELABELApolypeptides identified above. Fragments can comprise at least oneepitope. Methods of identifying epitopes are well known in the art.Fragments will typically comprise at least 5 amino acids, such as atleast 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or more aminoacids.

Included are fragments comprising or consisting of 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or more residuesfrom a relevant ELABELA amino acid sequence. In preferred embodiments ofthe aspects described herein, such ELABELA fragments comprise one ormore biological activities of a native ELABELA polypeptide, such asmaintaining self-renewal, pluripotency, or both of a stem cell, asdescribed herein.

We further describe peptides comprising a portion of a ELABELApolypeptide as described here. Thus, fragments of ELABELA and itshomologues, variants or derivatives are included. The peptides can bebetween 2 and 60 amino acids, such as between 4 and 50 amino acids inlength. The peptide can be derived from a ELABELA polypeptide asdisclosed here, for example by digestion with a suitable enzyme, such astrypsin. Alternatively the peptide, fragment, etc can be made byrecombinant means, or synthesised synthetically via chemical means, suchas solid phase synthesis.

Accordingly, provided herein in some aspects are methods of making anELABELA polypeptide or fragment thereof, the methods comprising:

(a) expressing a nucleic acid encoding a sequence comprisingCXXXRCXXXHSRVPFP (SEQ ID NO: 1) in a cell, wherein X is an/any aminoacid residue, and wherein the polypeptide fragment maintainsself-renewal, pluripotency, or both of a stem cell; or(b) using chemical synthesis to generate a synthetic polypeptide orfragment thereof comprising CXXXRCXXXHSRVPFP (SEQ ID NO: 1) or afragment having the sequence of SEQ ID NO: 53, wherein X is an/any aminoacid residue, and wherein the polypeptide fragment maintainsself-renewal, pluripotency, or both of a stem cell.

In some embodiments of the methods described herein, the cell expressingthe nucleic acid encoding a sequence comprising CXXXRCXXXHSRVPFP (SEQ IDNO: 1) is a bacterial, fungal, or yeast cell.

In some embodiments of the methods described herein, the sequencecomprising CXXXRCXXXHSRVPFP (SEQ ID NO: 1) is selected from the groupconsisting of SEQ ID NOs: 2-36.

In some embodiments of the methods described herein, the ELABELApolypeptide or fragment thereof further comprises a label. In someembodiments of the methods described herein, wherein the label is aradioisotope. In some embodiments of the methods described herein, theradioisotope is ¹²⁵I.

In some embodiments of the methods described herein, the polypeptide orfragment thereof is derivatized.

Such ELABELA fragments can be used to generate probes to preferentiallydetect ELABELA expression, for example, through antibodies generatedagainst such fragments. These antibodies would be expected to bindspecifically to ELABELA, and are useful in the methods of diagnosis andtreatment disclosed here.

ELABELA and its fragments, homologues, variants and derivatives, can bemade by recombinant means. However they can also be made by syntheticmeans using chemical techniques well known to skilled persons, such assolid phase synthesis. The proteins can also be produced as fusionproteins, for example to aid in extraction and purification. Examples offusion protein partners include glutathione-S-transferase (GST), 6×His(SEQ ID NO: 97), GAL4 (DNA binding and/or transcriptional activationdomains) and β-galactosidase. It can also be convenient to include aproteolytic cleavage site between the fusion protein partner and theprotein sequence of interest to allow removal of fusion proteinsequences. The fusion protein can be one which will not hinder thefunction of the protein of interest sequence. Proteins can also beobtained by purification of cell extracts from animal cells.

The ELABELA polypeptides, variants, homologues, fragments andderivatives disclosed here can be in a substantially isolated form. Itwill be understood that such polypeptides can be mixed with carriers ordiluents which will not interfere with the intended purpose of theprotein and still be regarded as substantially isolated. A ELABELAvariant, homologue, fragment or derivative can also be in asubstantially purified form, in which case it will generally comprisethe protein in a preparation in which more than 90%, e.g. 95%, 98% or99% of the protein in the preparation is a protein.

The ELABELA polypeptides, variants, homologues, fragments andderivatives disclosed here can be labelled with a revealing label. Therevealing label can be any suitable label which allows the polypeptide,etc to be detected. Suitable labels include radioisotopes, e.g. ¹²⁵I,enzymes, antibodies, polynucleotides and linkers such as biotin.Labelled polypeptides can be used in diagnostic procedures such asimmunoassays to determine the amount of a polypeptide in a sample.Polypeptides or labelled polypeptides can also be used in serological orcell-mediated immune assays for the detection of immune reactivity tosaid polypeptides in animals and humans using standard protocols.

A ELABELA polypeptides, variants, homologues, fragments and derivativesdisclosed here, optionally labelled, can also be fixed to a solid phase,for example the surface of an immunoassay well or dipstick. Suchlabelled and/or immobilised polypeptides can be packaged into kits in asuitable container along with suitable reagents, controls, instructionsand the like. Such polypeptides and kits can be used in methods ofdetection of antibodies to the polypeptides or their allelic or speciesvariants by immunoassay.

Immunoassay methods are well known in the art and will generallycomprise: (a) providing a polypeptide comprising an epitope bindable byan antibody against said protein; (b) incubating a biological samplewith said polypeptide under conditions which allow for the formation ofan antibody-antigen complex; and (c) determining whetherantibody-antigen complex comprising said polypeptide is formed.

The ELABELA polypeptides, variants, homologues, fragments andderivatives disclosed here can be used in in vitro or in vivo cellculture systems to study the role of their corresponding genes andhomologues thereof in cell function, including their function indisease. For example, truncated or modified polypeptides can beintroduced into a cell to disrupt the normal functions which occur inthe cell.

The polypeptides can be introduced into the cell by in situ expressionof the polypeptide from a recombinant expression vector (see elsewherein this document). The expression vector optionally carries an induciblepromoter to control the expression of the polypeptide.

The use of appropriate host cells, such as insect cells or mammaliancells, is expected to provide for such post-translational modifications(e.g. myristolation, glycosylation, truncation, lapidation and tyrosine,serine or threonine phosphorylation) as can be needed to confer optimalbiological activity on recombinant expression products.

Such cell culture systems in which the ELABELA polypeptides, variants,homologues, fragments and derivatives disclosed here are expressed canbe used in assay systems to identify candidate substances whichinterfere with or enhance the functions of the polypeptides in the cell.

ELABELA Polypeptide Activities

An ELABELA polypeptide according to this document can comprise one ormore activities, such as biological activities of a native ELABELApolypeptide, such as a human ELABELA polypeptide having SEQ ID NO: 20.Such activity can be referred to for convenience as an “ELABELAactivity” or an “ELABELA polypeptide activity”.

Where reference is made to the “activity” or “biological activity” of apolypeptide such as ELABELA, these terms are intended to refer to themetabolic or physiological function of ELABELA, including similaractivities or improved activities or these activities with decreasedundesirable side effects.

For example, an ELABELA activity can comprise any activity of a nativeELABELA polypeptide. It can comprise for example any physical,biochemical, enzymatic, biological etc activity of a native ELABELApolypeptide.

In particular, ELABELA activities of the polypeptide, fragments,variants, homologues, and derivatives described herein can include anyone or more of the following:

-   -   ability to maintain self-renewal of a stem cell    -   ability to maintain pluripotency of a stem cell    -   ability to inhibit apoptosis    -   ability to bind to the cell surface of an embryonic stem cell    -   ability to bind to apelin receptor (APLNR)    -   ability to bias differentiation of a stem cell toward an        endodermal or mesodermal lineage    -   cardioprotection, for example restoration or maintenance of        cardiac function during ischemia and/or reperfusion    -   reduction of oxidative stress    -   reduction of infarct size

The ELABELA activity can include any one or more of the ability tomaintain the growth potential of a stem cell, ability to maintain theclonogenicity of a stem cell and ability to maintain the survival of astem cell.

The ELABELA activity can include one or both of the ability to besecreted by an embryonic stem cell and ability to be taken-up by anembryonic stem cell

Also included are antigenic and immunogenic activities of ELABELA.Examples of such activities, and methods of assaying and quantifyingthese activities, are known in the art, and are described in detaillater in this section.

Maintenance of Self-Renewal

The ELABELA polypeptide can have a property of a native ELABELApolypeptide comprising the ability to maintain self-renewal of a cellsuch as a stem cell.

Maintenance of Pluripotency

The ELABELA polypeptide can have a property of a native ELABELApolypeptide comprising the ability to maintain pluripotency of a cellsuch as a stem cell.

Assays for Maintenance of Self-Renewal/Pluripotency

Assays for maintenance of self-renewal and/or pluripotency are describedin detail in the section below headed “MAINTENANCE OF SELF-RENEWAL ORPLURIPOTENCY”.

Inhibition of Apoptosis

The ELABELA polypeptide can have a property of a native ELABELApolypeptide comprising the ability to inhibit apoptosis. The ELABELApolypeptide can have a property of a native ELABELA polypeptidecomprising the ability to promote cell growth.

Assay for Promotion of Cell Growth/Inhibition of Apoptosis

Apoptosis inhibitory activity can be assayed as described in detail inExample 2 below.

Briefly, hESCs are dissociated into single cells using Accutase (StemCell Technologies) and plated in the presence of 10 μM Y-27632 (ROCKinhibitor) for 12 hours (Watanabe et al., 2007).

xCELLigence real time growth assays are performed, with 4000 cellsplated per well of an E-plate (ACEA Biosciences) with media changesevery 48 hours.

ELABELA polypeptide is added at 2.5 μM (or 10 μg/ml). Cell cycle studiesare performed with Click-iT EDU staining kit (Invitrogen) and byperforming a double thymidine block (2.5 mM thymidine; 16 hour block, 8hour release, 16 hour block) followed by DAPI staining for DNA contentat the indicated times following release.

Apoptosis assays are performed by plating control andDoxycycline-treated cells without Y-27632 onto matrigel for 6 hours,followed by harvesting and staining for Annexin V and activated Caspase3.

The ELABELA polypeptide can be such that a concentration of 10 μM ofELABELA polypeptide exposed to human ES cells over a period of 120 hoursincreases the cell index of the human ES cells by at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 100%, at least 150%, at least200% or more compared to control cells not exposed to ELABELApolypeptide.

The ELABELA polypeptide can be such that a concentration of 10 μM ofELABELA polypeptide exposed to human ES cells over a period of 120 hoursincreases the cell index of the human ES cells by 2.5 times or more, 3times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5times or more, 5.5 times or more, 6 times or more, 6.5 times or more, 7times or more, 7.5 times or more, 8 times or more, 8.5 times or more, 9times or more, 10 times or more, 15 times or more, 20 times or more, 30times or more or 40 times or more compared to control cells not exposedto ELABELA polypeptide.

By the term “cell index” we mean the quantitative measure (such as thenumber) of viable cells at any given time compared to the initial numberof cells seeded at the start of a cell culture assay (see Ke N, Wang X,Xu X, Abassi Y A. The xCELLigence system for real-time and label-freemonitoring of cell viability. Methods Mol Biol. 2011; 740:33-43).

ES Cell Surface Binding

The ELABELA polypeptide can have a property of a native ELABELApolypeptide comprising the ability to bind to the cell surface of an EScell.

Assay for ES Cell Surface Binding

ES cell surface binding activity assays can be performed with a numberof methods known to the person skilled in the art, for example asdescribed in Example 7 below.

ELABELA polypeptide can be exposed to embryonic stem cells such as hEScells and allowed to bind to their cognate cell surface receptors. hEScells can then be fixed and stained with an anti-ELABELA antibody, suchas any of the antibodies having ELABELA polypeptide binding activity, asdescribed in this document. Chromogenic methods to reveal antibodybinding are known to the art, and can include alkaline phosphatase,digoxigenin, based methods etc. Confocal microscopy can be used toreveal cell-surface binding (as opposed to intracellular binding).

Apelin Receptor (APLNR) Binding

The ELABELA polypeptide can have a property of a native ELABELApolypeptide comprising the ability to bind to an apelin receptor(APLNR).

Assay for Apelin Receptor Binding

Apelin receptor binding assays are known in the art and are described indetail in, for example, Angela Giddings, Scott Runyon, James Thomas,Julianne Tajuba, Katherine Bortoff, Rangan Maitra (2010). Development ofa functional HTS assay for the APJ receptor. International Journal ofHigh Throughput Screening. Volume 2010:1 Pages 39-47.

Biasing Towards Endo or Mesodermal Lineages

The ELABELA polypeptide can have a property of a native ELABELApolypeptide comprising the ability to bias, poise or steer a pluripotentcell toward an endodermal or mesodermal lineage. The ELABELA polypeptidecan poise a cell such as a stem cell towards the mesendoderm lineagewithout causing overt lineage commitment.

Assay for Ability to Bias Towards Endo or Mesodermal Lineages

Assays for mesoendodermal lineage bias are known in the art and aredescribed for example in International (PCT) Patent Publication NumberWO 2007/050043.

Assays for bias towards endodermal or mesodermal lineages can also beperformed as described for example in Example 3 and Example 20 below.

Any suitable ES cell culture, such as a human ES cell line Shef4 cellline (Inniss and Moore, 2006), can be used. ES cells are cultured in thepresence of a suitable concentration of ELABELA polypeptide (2.5 μM or10 μg/ml) for a suitable amount of time such as 24 hours, 48 hours, 72hours, 96 hours, 120 hours.

Following incubation, expression of mesendodermal markers includingGATA6, GATA4, FOXA2, EOMES and BRA can be assayed by any suitable means,for example using RT-PCR, qPCR or immunofluorescence, for example.

Thus, RNA can be extracted using RNeasy kit (Invitrogen). qPCR reactionscan be carried out using either Universal FastStart SYBR Green Mastermix(Roche) or using the Universal Probe Library system (Roche) in tandemwith Taqman Fast Mastermix (Invitrogen).

Primer sequences for each of these markers are known in the art and arealso listed in for example in Table E1 (Example 3).

Expression of cell surface markers such as SSEA3 and TRA-1-60 can beassayed to confirm that the ES cells do not lose stemness.

Cardioprotection

The ELABELA polypeptide can have a property of a native ELABELApolypeptide comprising cardioprotection. The cardioprotection cancomprise restoration or maintenance of cardiac function during ischemiaand/or reperfusion.

Cardioprotection can also be assayed as the ability to reduce infarctsize. Reduction of infarct can be assayed in a mouse or pig model ofmyocardial ischemia and reperfusion injury.

Assay for Cardioprotection

Cardioprotection can for example be assayed using any one or more of themethods described in Examples 5, 10, 14 and 20 of International (PCT)Patent Publication WO 2009/105044.

Oxidative Stress

The ELABELA polypeptide can have a property of a native ELABELApolypeptide comprising the ability to reduce oxidative stress (orcytoprotection).

Assay for Oxidative Stress

The reduction of oxidative stress can for example be assayed using an invitro assay of hydrogen peroxide (H₂O₂)-induced cell death. In summary,hydrogen peroxide (H₂O₂)-mediated oxidative stress is induced in humanleukemic CEM cells and cell viability is monitored by Trypanblue-exclusion. Human leukemic CEM cells are incubated with ELABELApolypeptide (with saline as a control) and treated with 50 μM H₂O₂ toinduce oxidative stress. Cell viability is assessed using Trypan Blueexclusion at 12, 24, 36 and 48 hours after H₂O₂ treatment.

The reduction of oxidative stress can further be assayed using an invivo assay of DNA oxidation. In vivo oxidative stress can also beassayed as follows. Pigs are treated with the ELABELA polypeptide (withsaline as a control). Tissue sections of pig heart are obtained. Nuclearoxidative stress in tissue sections of treated and untreated pigs isquantified by 8-OHdG immunostaining for oxidized DNA. The tissuesections are assayed for intense nuclear staining indicative of DNAoxidation and oxidative stress.

Reduction of Infarct Size

The ELABELA polypeptide can have a property of a native ELABELApolypeptide comprising the ability to reduce infarct size.

Assay for Infarct Size

Infarct size can for example be assayed using any one or more of themethods described in Examples 6 and 13 of International (PCT) PatentPublication WO 2009/105044.

ELABELA Nucleic Acids

The methods and compositions described here can make use of ELABELApolynucleotides, ELABELA nucleotides and ELABELA nucleic acids, as wellas variants, homologues, derivatives and fragments of any of these.

The terms “ELABELA polynucleotide”, “ELABELA nucleotide” and “ELABELAnucleic acid” can be used interchangeably, and should be understood tospecifically include both cDNA and genomic ELABELA sequences. Theseterms are also intended to include a nucleic acid sequence capable ofencoding a ELABELA polypeptide and/or a fragment, derivative, homologueor variant of this. These terms are also intended to include a nucleicacid sequence which is a fragment, derivative, homologue or variant ofan ELABELA polypeptide having a specific sequence disclosed in thisdocument, for example as set out in the sequence listings.

Where reference is made to a ELABELA nucleic acid, this should be takenas a reference to a nucleic acid sequence capable of encoding an ELABELApolypeptide. In preferred embodiments of the aspects described herein,such nucleic acids encode ELABELA polypeptides comprising one or morebiological activities of a native ELABELA polypeptide, such asmaintaining self-renewal, pluripotency, or both of a stem cell, asdescribed herein.

For example, an ELABELA nucleic acid sequence can be capable of encodinga polypeptide comprising a sequence CXXXRCXXXHSRVPFP (SEQ ID NO: 1), inwhich X signifies an amino acid residue. The resulting encodedpolypeptide sequence can comprise ELABELA activity, such as beingcapable of maintaining self-renewal and/or pluripotency of a stem cell.

An ELABELA nucleic acid can also be taken generally to refer to anymember of the ELABELA family of nucleic acids.

ELABELA nucleic acids can for example be capable of encodingpolypeptides comprising any of the sequences set out as SEQ ID NO: 2 toSEQ ID NO: 19. An ELABELA nucleic acid can be capable of encoding apolypeptide comprising a sequence CLQRRCMPLHSRVPFP (SEQ ID NO: 60).

Examples of ELABELA nucleic acids include those selected from the groupconsisting of SEQ ID NO: 37 to SEQ ID NO: 41 or SEQ ID NO: 42 to SEQ IDNO: 46. For example, a human ELABELA nucleic acid sequence having thesequence SEQ ID NO: 37 is disclosed.

Also included are any one or more of the nucleic acid sequences set outas “Other ELABELA nucleic acid sequences” elsewhere in this document.

For example, the ELABELA nucleic acid can comprise a human ELABELAsequence SEQ ID NO: 37.

ELABELA nucleic acids can be used for a variety of means, as describedin this document. For example, ELABELA nucleic acids can be used treatan individual suffering from, or suspected to be suffering from anELABELA associated condition, or to prevent such a condition or toalleviate any symptoms arising as a result of such a condition. They canbe used to maintain or sustain pluripotency or self-renewal or both of acell such as a stem cell. ELABELA nucleic acids can also be used for theexpression or production of ELABELA polypeptides. Other uses will beevident to the skilled reader, and are also encompassed in thisdocument.

The term “polynucleotide”, as used in this document, generally refers toany polyribonucleotide or polydeoxribonucleotide, which can beunmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include,without limitation single- and double-stranded DNA, DNA that is amixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single-anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatcan be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, “polynucleotide”refers to triple-stranded regions comprising RNA or DNA or both RNA andDNA. The term polynucleotide also includes DNAs or RNAs containing oneor more modified bases and DNAs or RNAs with backbones modified forstability or for other reasons. “Modified” bases include, for example,tritylated bases and unusual bases such as inosine. A variety ofmodifications has been made to DNA and RNA; thus, “polynucleotide”embraces chemically, enzymatically or metabolically modified forms ofpolynucleotides as typically found in nature, as well as the chemicalforms of DNA and RNA characteristic of viruses and cells.“Polynucleotide” also embraces relatively short polynucleotides, oftenreferred to as oligonucleotides.

It will be understood by the skilled person that numerous nucleotidesequences can encode the same polypeptide as a result of the degeneracyof the genetic code.

As used herein, the term “nucleotide sequence” refers to nucleotidesequences, oligonucleotide sequences, polynucleotide sequences andvariants, homologues, fragments and derivatives thereof (such asportions thereof). The nucleotide sequence can be DNA or RNA of genomicor synthetic or recombinant origin which can be double-stranded orsingle-stranded whether representing the sense or antisense strand orcombinations thereof. The term nucleotide sequence can be prepared byuse of recombinant DNA techniques (for example, recombinant DNA).

The term “nucleotide sequence” can mean DNA.

Other Nucleic Acids

We also provide nucleic acids which are fragments, homologues, variantsor derivatives of ELABELA nucleic acids. The terms “variant”,“homologue”, “derivative” or “fragment” in relation to ELABELA nucleicacid include any substitution of, variation of, modification of,replacement of, deletion of or addition of one (or more) nucleic acidsfrom or to the sequence of a ELABELA nucleotide sequence. Unless thecontext admits otherwise, references to “ELABELA” and “ELABELA nucleicacid”, “ELABELA nucleotide sequence” etc include references to suchvariants, homologues, derivatives and fragments of ELABELA.

The resultant nucleotide sequence can encode a polypeptide having anyone or more ELABELA activity. The term “homologue” may be intended tocover identity with respect to structure and/or function such that theresultant nucleotide sequence encodes a polypeptide which has ELABELAactivity. For example, a homologue etc of ELABELA may have a increasedexpression level in cells from an individual suffering from an ELABELAassociated condition compared to normal cells. With respect to sequenceidentity (i.e. similarity), there may be at least 70%, at least 75%, atleast 85% or at least 90% sequence identity. There may be at least 95%,such as at least 98%, sequence identity to a relevant sequence such asany nucleic acid sequence shown in the sequence listings (e.g., aELABELA sequence having SEQ ID NO: 37). These terms also encompassallelic variations of the sequences.

Variants, Derivatives and Homologues

ELABELA nucleic acid variants, fragments, derivatives and homologues maycomprise DNA or RNA. They can be single-stranded or double-stranded.They can also be polynucleotides which include within them synthetic ormodified nucleotides. A number of different types of modification tooligonucleotides are known in the art. These include methylphosphonateand phosphorothioate backbones, addition of acridine or polylysinechains at the 3′ and/or 5′ ends of the molecule. For the purposes ofthis document, it is to be understood that the polynucleotides can bemodified by any method available in the art. Such modifications can becarried out in order to enhance the in vivo activity or life span ofpolynucleotides of interest.

Where the polynucleotide is double-stranded, both strands of the duplex,either individually or in combination, are encompassed by the methodsand compositions described here. Where the polynucleotide issingle-stranded, it is to be understood that the complementary sequenceof that polynucleotide is also included.

The terms “variant”, “homologue” or “derivative” in relation to anucleotide sequence include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence. Said variant, homologues orderivatives can code for a polypeptide having biological activity. Suchfragments, homologues, variants and derivatives of ELABELA can comprisemodulated activity, as set out above.

As indicated above, with respect to sequence identity, a “homologue” canhave at least 5% identity, at least 10% identity, at least 15% identity,at least 20% identity, at least 25% identity, at least 30% identity, atleast 35% identity, at least 40% identity, at least 45% identity, atleast 50% identity, at least 55% identity, at least 60% identity, atleast 65% identity, at least 70% identity, at least 75% identity, atleast 80% identity, at least 85% identity, at least 90% identity, or atleast 95% identity to the relevant sequence, such as any nucleic acidsequence shown in the sequence listings (e.g., a ELABELA sequence havingSEQ ID NO: 37).

There can be at least 95% identity, at least 96% identity, at least 97%identity, at least 98% identity or at least 99% identity. Nucleotideidentity comparisons can be conducted as described above. A sequencecomparison program which can be used is the GCG Wisconsin Bestfitprogram described above. The default scoring matrix has a match value of10 for each identical nucleotide and −9 for each mismatch. The defaultgap creation penalty is −50 and the default gap extension penalty is −3for each nucleotide.

Hybridisation

We further describe nucleotide sequences that are capable of hybridisingselectively to any of the sequences presented herein, or any variant,fragment or derivative thereof, or to the complement of any of theabove. Nucleotide sequences can be at least 5, 10, or 15 nucleotides inlength, such as at least 20, 30, 40 or 50 nucleotides in length.

The term “hybridization” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” as well as the process of amplification as carried out inpolymerase chain reaction technologies.

Polynucleotides capable of selectively hybridising to the nucleotidesequences presented herein, or to their complement, can be at least 40%homologous, at least 45% homologous, at least 50% homologous, at least55% homologous, at least 60% homologous, at least 65% homologous, atleast 70% homologous, at least 75% homologous, at least 80% homologous,at least 85% homologous, at least 90% homologous, or at least 95%homologous to the corresponding nucleotide sequences presented herein,such as any nucleic acid sequence shown in the sequence listings (e.g.,a ELABELA sequence having SEQ ID NO: 37). Such polynucleotides can begenerally at least 70%, at least 80 or 90% or at least 95% or 98%homologous to the corresponding nucleotide sequences over a region of atleast 5, 10, 15 or 20, such as at least 25 or 30, for instance at least40, 60 or 100 or more contiguous nucleotides.

The term “selectively hybridizable” means that the polynucleotide usedas a probe is used under conditions where a target polynucleotide isfound to hybridize to the probe at a level significantly abovebackground. The background hybridization can occur because of otherpolynucleotides present, for example, in the cDNA or genomic DNA librarybeing screening. In this event, background implies a level of signalgenerated by interaction between the probe and a non-specific DNA memberof the library which is less than 10 fold, such as less than 100 fold asintense as the specific interaction observed with the target DNA. Theintensity of interaction can be measured, for example, by radiolabellingthe probe, e.g. with ³²P or ³³P or with non-radioactive probes (e.g.,fluorescent dyes, biotin or digoxigenin).

Hybridization conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152,Academic Press, San Diego Calif.), and confer a defined “stringency” asexplained elsewhere in this document.

Maximum stringency typically occurs at about Tm-5° C. (5° C. below theTm of the probe); high stringency at about 5° C. to 10° C. below Tm;intermediate stringency at about 10° C. to 20° C. below Tm; and lowstringency at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identify or detect identical polynucleotide sequences while anintermediate (or low) stringency hybridization can be used to identifyor detect similar or related polynucleotide sequences.

We provide nucleotide sequences that can be able to hybridise to theELABELA nucleic acids, fragments, variants, homologues or derivativesunder stringent conditions (e.g. 65° C. and 0.1×SSC (1×SSC=0.15 M NaCl,0.015 M Na₃ Citrate pH 7.0)).

Generation of Homologues, Variants and Derivatives

Polynucleotides which are not 100% identical to the relevant sequences(e.g., a human ELABELA sequence having SEQ ID NO: 37) but which are alsoincluded, as well as homologues, variants and derivatives of ELABELA canbe obtained in a number of ways. Other variants of the sequences can beobtained for example by probing DNA libraries made from a range ofindividuals, for example individuals from different populations. Forexample, ELABELA homologues can be identified from other individuals, orother species. Further recombinant ELABELA nucleic acids andpolypeptides can be produced by identifying corresponding positions inthe homologues, and synthesising or producing the molecule as describedelsewhere in this document.

In addition, other viral/bacterial, or cellular homologues of ELABELA,particularly cellular homologues found in mammalian cells (e.g. rat,mouse, bovine and primate cells), can be obtained and such homologuesand fragments thereof in general will be capable of selectivelyhybridising to human ELABELA. Such homologues can be used to designnon-human ELABELA nucleic acids, fragments, variants and homologues.Mutagenesis can be carried out by means known in the art to producefurther variety.

Sequences of ELABELA homologues can be obtained by probing cDNAlibraries made from or genomic DNA libraries from other animal species,and probing such libraries with probes comprising all or part of any ofthe ELABELA nucleic acids, fragments, variants and homologues, or otherfragments of ELABELA under conditions of medium to high stringency.

Similar considerations apply to obtaining species homologues and allelicvariants of the polypeptide or nucleotide sequences disclosed here.

Variants and strain/species homologues can also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences within the sequences of the ELABELA nucleic acids. Conservedsequences can be predicted, for example, by aligning the amino acidsequences from several variants/homologues. Sequence alignments can beperformed using computer software known in the art. For example the GCGWisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degeneratepositions and will be used at stringency conditions lower than thoseused for cloning sequences with single sequence primers against knownsequences. It will be appreciated by the skilled person that overallnucleotide homology between sequences from distantly related organismsis likely to be very low and thus in these situations degenerate PCR canbe the method of choice rather than screening libraries with labelledfragments the ELABELA sequences.

In addition, homologous sequences can be identified by searchingnucleotide and/or protein databases using search algorithms such as theBLAST suite of programs.

Alternatively, such polynucleotides can be obtained by site directedmutagenesis of characterised sequences, for example, ELABELA nucleicacids, or variants, homologues, derivatives or fragments thereof. Thiscan be useful where for example silent codon changes are required tosequences to optimise codon preferences for a particular host cell inwhich the polynucleotide sequences are being expressed. Other sequencechanges can be desired in order to introduce restriction enzymerecognition sites, or to alter the property or function of thepolypeptides encoded by the polynucleotides.

The polynucleotides described here can be used to produce a primer, e.g.a PCR primer, a primer for an alternative amplification reaction, aprobe e.g. labelled with a revealing label by conventional means usingradioactive or non-radioactive labels, or the polynucleotides can becloned into vectors. Such primers, probes and other fragments will be atleast 8, 9, 10, or 15, such as at least 20, for example at least 25, 30or 40 nucleotides in length, and are also encompassed by the term“polynucleotides” as used herein.

Polynucleotides such as a DNA polynucleotides and probes can be producedrecombinantly, synthetically, or by any means available to those ofskill in the art. They can also be cloned by standard techniques.

In general, primers will be produced by synthetic means, involving astep wise manufacture of the desired nucleic acid sequence onenucleotide at a time. Techniques for accomplishing this using automatedtechniques are readily available in the art.

Primers comprising fragments of ELABELA are particularly useful in themethods of detection of ELABELA expression, such as up-regulation ofELABELA expression, for example, as associated with an ELABELAassociated condition. Suitable primers for amplification of ELABELA canbe generated from any suitable stretch of ELABELA. Primers which can beused include those capable of amplifying a sequence of ELABELA which isspecific.

Although ELABELA primers can be provided on their own, they are mostusefully provided as primer pairs, comprising a forward primer and areverse primer.

Longer polynucleotides will generally be produced using recombinantmeans, for example using a PCR (polymerase chain reaction) cloningtechniques. This will involve making a pair of primers (e.g. of about 15to 30 nucleotides), bringing the primers into contact with mRNA or cDNAobtained from an animal or human cell, performing a polymerase chainreaction under conditions which bring about amplification of the desiredregion, isolating the amplified fragment (e.g. by purifying the reactionmixture on an agarose gel) and recovering the amplified DNA. The primerscan be designed to contain suitable restriction enzyme recognition sitesso that the amplified DNA can be cloned into a suitable cloning vector

Polynucleotides or primers can carry a revealing label. Suitable labelsinclude radioisotopes such as ³²P or ³⁵S, digoxigenin, fluorescent dyes,enzyme labels, or other protein labels such as biotin. Such labels canbe added to polynucleotides or primers and can be detected using bytechniques known per se. Polynucleotides or primers or fragments thereoflabelled or unlabeled can be used by a person skilled in the art innucleic acid-based tests for detecting or sequencing polynucleotides inthe human or animal body.

Such tests for detecting generally comprise bringing a biological samplecontaining DNA or RNA into contact with a probe comprising apolynucleotide or primer under hybridising conditions and detecting anyduplex formed between the probe and nucleic acid in the sample. Suchdetection can be achieved using techniques such as PCR or byimmobilising the probe on a solid support, removing nucleic acid in thesample which is not hybridised to the probe, and then detecting nucleicacid which has hybridised to the probe. Alternatively, the samplenucleic acid can be immobilised on a solid support, and the amount ofprobe bound to such a support can be detected. Suitable assay methods ofthis and other formats can be found in for example WO89/03891 andWO90/13667.

Tests for sequencing nucleotides, for example, the ELABELA nucleicacids, involve bringing a biological sample containing target DNA or RNAinto contact with a probe comprising a polynucleotide or primer underhybridising conditions and determining the sequence by, for example theSanger dideoxy chain termination method (see Sambrook et al.).

Such a method generally comprises elongating, in the presence ofsuitable reagents, the primer by synthesis of a strand complementary tothe target DNA or RNA and selectively terminating the elongationreaction at one or more of an A, C, G or T/U residue; allowing strandelongation and termination reaction to occur; separating out accordingto size the elongated products to determine the sequence of thenucleotides at which selective termination has occurred. Suitablereagents include a DNA polymerase enzyme, the deoxynucleotides dATP,dCTP, dGTP and dTTP, a buffer and ATP. Dideoxynucleotides are used forselective termination.

ELABELA Control Regions

For some purposes, it can be necessary to utilise or investigate controlregions of ELABELA. Such control regions include promoters, enhancersand locus control regions. By a control region we mean a nucleic acidsequence or structure which is capable of modulating the expression of acoding sequence which is operatively linked to it.

For example, control regions are useful in generating transgenic animalsexpressing ELABELA. Furthermore, control regions can be used to generateexpression constructs for ELABELA. This is described in further detailelsewhere in this document.

Identification of control regions of ELABELA is straightforward, and canbe carried out in a number of ways. For example, the coding sequence ofELABELA can be obtained from an organism, by screening a cDNA libraryusing a human or mouse ELABELA cDNA sequence as a probe. 5′ sequencescan be obtained by screening an appropriate genomic library, or byprimer extension as known in the art. Database searching of genomedatabases can also be employed. Such 5′ sequences which are particularlyof interest include non-coding regions. The 5′ regions can be examinedby eye, or with the aid of computer programs, to identify sequencemotifs which indicate the presence of promoter and/or enhancer regions.

Furthermore, sequence alignments can be conducted of ELABELA nucleicacid sequences from two or more organisms. By aligning ELABELA sequencesfrom different species, it is possible to determine which regions of theamino acid sequence are conserved between different species. Suchconserved regions are likely to contain control regions for the gene inquestion (i.e., ELABELA). The mouse and human genomic sequences asdisclosed here, for example, a mouse ELABELA genomic sequence, can beemployed for this purpose. Furthermore, ELABELA homologues from otherorganisms can be obtained using standard methods of screening usingappropriate probes generated from the mouse and human ELABELA sequences.The genome of the pufferfish (Takifugu rubripes) or zebrafish can alsobe screened to identify a ELABELA homologue; thus, several zebrafishsequences of ELABELA have been identified (noted above). Comparison ofthe 5′ non-coding region of the Fugu or zebrafish ELABELA gene with amouse or human genomic ELABELA sequence can be used to identifyconserved regions containing control regions.

Deletion studies can also be conducted to identify promoter and/orenhancer regions for ELABELA.

The identity of putative control regions can be confirmed by molecularbiology experiments, in which the candidate sequences are linked to areporter gene and the expression of the reporter detected.

Modulation of ELABELA Expression

We describe a method of manipulating a cell, the method comprisingmodulating, such as up-regulating, any combination of the expression,amount or activity of an ELABELA polypeptide in or of the cell. Themethod can comprise exposing the cell to an ELABELA polypeptide.

Alternatively, or in addition, ELABELA expression, amount or activitycan be up-regulated by introducing an ELABELA expression construct (alsoknown as an ELABELA construct) into the cell. The ELABELA expressionconstruct or ELABELA construct can comprise an ELABELA nucleic acid or avector (such as an expression vector) comprising an ELABELA nucleic acidsequence.

Mechanisms for delivery of such constructs, nucleic acids and vectorscan comprise electroporation, calcium phosphate transformation orparticle bombardment. However, transfer of the construct can beperformed by any of the methods mentioned which physically or chemicallypermeabilize the cell membrane.

1. Electroporation

The ELABELA construct can be introduced into the cells viaelectroporation. Electroporation involves the exposure of a suspensionof cells and DNA to a high-voltage electric discharge.

Transfection of eukaryotic cells using electroporation has been quitesuccessful. Mouse pre-B lymphocytes have been transfected with humankappa-immunoglobulin genes (Potter et al., 1984), and rat hepatocyteshave been transfected with the chloramphenicol acetyltransferase gene(Tur-Kaspa et al., 1986) in this manner.

It is contemplated that electroporation conditions for cells fromdifferent sources can be optimized. One can particularly with tooptimize such parameters as the voltage, the capacitance, the time andthe electroporation media composition. The execution of other routineadjustments will be known to those of skill in the art.

2. Particle Bombardment

One of the ways of transferring a naked DNA construct into cellsinvolves particle bombardment. This method depends on the ability toaccelerate DNA-coated microprojectiles to a high velocity allowing themto pierce cell membranes and enter cells without killing them (Klein etal., 1987). The microprojectiles used have consisted of biologicallyinert substances such as tungsten, platinum or gold beads.

It is contemplated that in some instances DNA precipitation onto metalparticles would not be necessary for DNA delivery to a recipient cellusing particle bombardment. It is contemplated that particles cancontain DNA rather than be coated with DNA. Hence it is proposed thatDNA-coated particles can increase the level of DNA delivery via particlebombardment but are not, in and of themselves, necessary.

Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force (Yang etal., 1990). Another method involves the use of a Biolistic ParticleDelivery System, which can be used to propel particles coated with DNAthrough a screen, such as stainless steel or Nytex screen, onto a filtersurface covered with cells in suspension. The screen disperses theparticles so that they are not delivered to the recipient cells in largeaggregates. It is believed that a screen intervening between theprojectile apparatus and the cells to be bombarded reduces the size ofprojectile aggregates and can contribute to a higher frequency oftransformation by reducing the damage inflicted on the recipient cellsby projectiles that are too large.

For the bombardment, cells in suspension can be concentrated on filters,or alternatively on solid culture medium. The cells to be bombarded arepositioned at an appropriate distance below the macroprojectile stoppingplate. If desired, one or more screens are also positioned between theacceleration device and the cells to be bombarded.

In bombardment transformation, one can optimize the prebombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity or either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of primordial germ cells.

Accordingly, it is contemplated that one can wish to adjust various ofthe bombardment parameters in small scale studies to fully optimize theconditions. One can particularly wish to adjust physical parameters suchas gap distance, flight distance, tissue distance and helium pressure.One can also optimize the trauma reduction factors by modifyingconditions which influence the physiological state of the recipientcells and which can therefore influence transformation and integrationefficiencies. For example, the osmotic state, tissue hydration and thesubculture stage or cell cycle of the recipient cells can be adjustedfor optimum transformation. The execution of other routine adjustmentswill be known to those of skill in the art.

3. Viral Transformation Adenoviral Infection

One method for delivery of the ELABELA nucleic acid constructs involvesthe use of an adenovirus expression vector. Although adenovirus vectorsare known to have a low capacity for integration into genomic DNA, thisfeature is counterbalanced by the high efficiency of gene transferafforded by these vectors. “Adenovirus expression vector” is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and (b) to ultimately express aconstruct that has been cloned therein.

The vector comprises a genetically engineered form of adenovirus.Knowledge of the genetic organization or adenovirus, a 36 kb, linear,double-stranded DNA virus, allows substitution of large pieces ofadenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz,1992). In contrast to retrovirus, the adenoviral infection of host cellsdoes not result in chromosomal integration because adenoviral DNA canreplicate in an episomal manner without potential genotoxicity. Also,adenoviruses are structurally stable, and no genome rearrangement hasbeen detected after extensive amplification.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget-cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNA's issued from this promoterpossess a 5′-tripartite leader (TPL) sequence which makes them suitablemRNA's for translation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus can be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones and Shenk, 1978), the current adenovirus vectors, with the helpof 293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kb of DNA. Combined with theapproximately 5.5 kb of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kb, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone.

Helper cell lines can be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells can be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. A suitablehelper cell line is 293.

Racher et al. (1995) disclosed improved methods for culturing 293 cellsand propagating adenovirus. In one format, natural cell aggregates aregrown by inoculating individual cells into 1 liter siliconized spinnerflasks (Techne, Cambridge, UK) containing 100-200 ml of medium.Following stirring at 40 rpm, the cell viability is estimated withtrypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin,Stone, UK) (5 g/l) is employed as follows. A cell inoculum, resuspendedin 5 ml of medium, is added to the carrier (50 ml) in a 250 mlErlenmeyer flask and left stationary, with occasional agitation, for 1to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the methods and compositions described here. The adenoviruscan be of any of the 42 different known serotypes or subgroups A-F.Adenovirus type 5 of subgroup C can be used as starting material inorder to obtain the conditional replication-defective adenovirus vectorfor use in the methods and compositions described here. This is becauseAdenovirus type 5 is a human adenovirus about which a great deal ofbiochemical and genetic information is known, and it has historicallybeen used for most constructions employing adenovirus as a vector.

As stated above, the typical vector according to the methods andcompositions described hereis replication defective and will not have anadenovirus E1 region. Thus, it will be most convenient to introduce thetransforming construct at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to the methodsand compositions described here. The polynucleotide encoding the gene ofinterest can also be inserted in lieu of the deleted E3 region in E3replacement vectors as described by Karlsson et al. (1986) or in the E4region where a helper cell line or helper virus complements the E4defect.

Adenovirus growth and manipulation is known to those of skill in theart, and exhibits broad host range in vitro and in vivo. This group ofviruses can be obtained in high titers, e.g., 10.sup.9-10.sup.11plaque-forming units per ml, and they are highly infective. The lifecycle of adenovirus does not require integration into the host cellgenome. The foreign genes delivered by adenovirus vectors are episomaland, therefore, have low genotoxicity to host cells. No side effectshave been reported in studies of vaccination with wild-type adenovirus(Couch et al., 1963; Top et al., 1971), demonstrating their safety andtherapeutic potential as in vivo gene transfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

AAV Infection

Adeno-associated virus (AAV) is an attractive vector system for use inthe methods and compositions described hereas it has a high frequency ofintegration and it can infect nondividing cells, thus making it usefulfor delivery of genes into mammalian cells in tissue culture (Muzyczka,1992). AAV has a broad host range for infectivity (Tratschin, et al.,1984; Laughlin, et al., 1986; Lebkowski, et al., 1988; McLaughlin, etal., 1988), which means it is applicable for use with the methods andcompositions described here. Details concerning the generation and useof rAAV vectors are described in U.S. Pat. No. 5,139,941 and U.S. Pat.No. 4,797,368, each incorporated herein by reference.

Studies demonstrating the use of AAV in gene delivery include LaFace etal. (1988); Zhou et al. (1993); Flotte et al. (1993); and Walsh et al.(1994). Recombinant AAV vectors have been used successfully for in vitroand in vivo transduction of marker genes (Kaplitt, et al., 1994;Lebkowski, et al., 1988; Samulski, et al., 1989; Shelling and Smith,1994; Yoder, et al., 1994; Zhou, et al., 1994; Hermonat and Muzyczka,1984; Tratschin, et al., 1985; McLaughlin, et al., 1988) and genesinvolved in human diseases (Flotte, et al., 1992; Luo, et al., 1994;Ohi, et al., 1990; Walsh, et al., 1994; Wei, et al., 1994). Recently, anAAV vector has been approved for phase I human trials for the treatmentof cystic fibrosis.

AAV is a dependent parvovirus in that it requires coinfection withanother virus (either adenovirus or a member of the herpes virus family)to undergo a productive infection in cultured cells (Muzyczka, 1992). Inthe absence of coinfection with helper virus, the wild type AAV genomeintegrates through its ends into human chromosome 19 where it resides ina latent state as a provirus (Kotin et al., 1990; Samulski et al.,1991). rAAV, however, is not restricted to chromosome 19 for integrationunless the AAV Rep protein is also expressed (Shelling and Smith, 1994).When a cell carrying an AAV provirus is superinfected with a helpervirus, the AAV genome is “rescued” from the chromosome or from arecombinant plasmid, and a normal productive infection is established(Samulski, et al., 1989; McLaughlin, et al., 1988; Kotin, et al., 1990;Muzyczka, 1992).

Typically, recombinant AAV (rAAV) virus is made by cotransfecting aplasmid containing the gene of interest flanked by the two AAV terminalrepeats (McLaughlin et al., 1988; Samulski et al., 1989; eachincorporated herein by reference) and an expression plasmid containingthe wild type AAV coding sequences without the terminal repeats, forexample pIM45 (McCarty et al., 1991; incorporated herein by reference).The cells are also infected or transfected with adenovirus or plasmidscarrying the adenovirus genes required for AAV helper function. rAAVvirus stocks made in such fashion are contaminated with adenovirus whichmust be physically separated from the rAAV particles (for example, bycesium chloride density centrifugation). Alternatively, adenovirusvectors containing the AAV coding regions or cell lines containing theAAV coding regions and some or all of the adenovirus helper genes couldbe used (Yang et al., 1994a; Clark et al., 1995). Cell lines carryingthe rAAV DNA as an integrated provirus can also be used (Flotte et al.,1995).

Retroviral Infection

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

Concern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which the intactsequence from the recombinant virus inserts upstream from the gag, pol,env sequence integrated in the host cell genome. However, new packagingcell lines are now available that should greatly decrease the likelihoodof recombination (Markowitz et al., 1988; Hersdorffer et al., 1990).

Other Viral Vectors

Other viral vectors can be employed as ELABELA constructs in the methodsand compositions described here. Vectors derived from viruses such asvaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988) and herpesviruses can be employed. They offer severalattractive features for various mammalian cells (Friedmann, 1989;Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwichet al., 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. Chang et al. recently introduced thechloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virusgenome in the place of the polymerase, surface, and pre-surface codingsequences. It was cotransfected with wild-type virus into an avianhepatoma cell line. Culture media containing high titers of therecombinant virus were used to infect primary duckling hepatocytes.Stable CAT gene expression was detected for at least 24 days aftertransfection (Chang et al., 1991).

In still further embodiments, the ELABELA nucleic acids to be deliveredare housed within an infective virus that has been engineered to expressa specific binding ligand. The virus particle will thus bindspecifically to the cognate receptors of the target cell and deliver thecontents to the cell. A novel approach designed to allow specifictargeting of retrovirus vectors was recently developed based on thechemical modification of a retrovirus by the chemical addition oflactose residues to the viral envelope. This modification can permit thespecific infection of hepatocytes via sialoglycoprotein receptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et al.,1989). Using antibodies against major histocompatibility complex class Iand class II antigens, they demonstrated the infection of a variety ofhuman cells that bore those surface antigens with an ecotropic virus invitro (Roux et al., 1989).

4. Calcium Phosphate Co-Precipitation or DEAE-Dextran Treatment

In other embodiments, the ELABELA nucleic acid construct is introducedto the cells using calcium phosphate co-precipitation. Mouse primordialgerm cells have been transfected with the SV40 large T antigen, withexcellent results (Watanabe et al., 1997). Human KB cells have beentransfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) usingthis technique. Also in this manner, mouse L(A9), mouse C127, CHO, CV-1,BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene(Chen and Okayama, 1987), and rat hepatocytes were transfected with avariety of marker genes (Rippe et al., 1990).

In another embodiment, the ELABELA expression construct is deliveredinto the cell using DEAE-dextran followed by polyethylene glycol. Inthis manner, reporter plasmids were introduced into mouse myeloma anderythroleukemia cells (Gopal, 1985).

5. Direct Microinjection or Sonication Loading

Further embodiments include the introduction of the ELABELA nucleic acidconstruct by direct microinjection or sonication loading. Directmicroinjection has been used to introduce ELABELA nucleic acidconstructs into Xenopus oocytes (Harland and Weintraub, 1985), andLTK.sup.-fibroblasts have been transfected with the thymidine kinasegene by sonication loading (Fechheimer et al., 1987).

6. Liposome Mediated Transformation

In a further embodiment, the ELABELA nucleic acid construct can beentrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated is an ELABELA nucleic acid constructcomplexed with Lipofectamine (Gibco BRL).

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful (Nicolau and Sene, 1982; Fraley et al.,1979; Nicolau et al., 1987). Wong et al. (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells.

In certain embodiments, the liposome can be complexed with ahemagglutinating virus (HVJ). This has been shown to facilitate fusionwith the cell membrane and promote cell entry of liposome-encapsulatedDNA (Kaneda et al., 1989). In other embodiments, the liposome can becomplexed or employed in conjunction with nuclear non-histonechromosomal proteins (HMG-1) (Kato et al., 1991). In yet furtherembodiments, the liposome can be complexed or employed in conjunctionwith both HVJ and HMG-1.

7. Adenoviral Assisted Transfection

In certain embodiments, the ELABELA nucleic acid construct is introducedinto the cell using adenovirus assisted transfection. Increasedtransfection efficiencies have been reported in cell systems usingadenovirus coupled systems (Kelleher and Vos, 1994; Cotten et al., 1992;Curiel, 1994), and the inventors contemplate using the same technique toincrease transfection efficiencies.

8. Receptor Mediated Transfection

Still further ELABELA constructs that can be employed to deliver theELABELA nucleic acid construct to the target cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis that will be occurringin the target cells. In view of the cell type-specific distribution ofvarious receptors, this delivery method adds a degree of specificity.Specific delivery in the context of another mammalian cell type isdescribed by Wu and Wu (1993; incorporated herein by reference).

Certain nucleic acid delivery constructs comprise a cellreceptor-specific ligand and a DNA-binding agent. Others comprise a cellreceptor-specific ligand to which the DNA construct to be delivered hasbeen operatively attached. Several ligands have been used forreceptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al., 1990;Ferkol et al., 1993; Perales et al., 1994; Myers, EPO 0273085), whichestablishes the operability of the technique.

In other embodiments, the DNA delivery vehicle component can comprise aspecific binding ligand in combination with a liposome. The nucleicacids to be delivered are housed within the liposome and the specificbinding ligand is functionally incorporated into the liposome membrane.The liposome will thus specifically bind to the receptors of the targetcell and deliver the contents to the cell. Such systems have been shownto be fimctional using systems in which, for example, epidermal growthfactor (EGF) is used in the receptor-mediated delivery of a nucleic acidto cells that exhibit upregulation of the EGF receptor.

In still further embodiments, the DNA delivery vehicle component of thedelivery vehicles can be a liposome itself, which can comprise one ormore lipids or glycoproteins that direct cell-specific binding. Forexample, Nicolau et al. (1987) employed lactosyl-ceramide, agalactose-terminal asialoganglioside, incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes.It is contemplated that the ELABELA nucleic acid described here can bespecifically delivered into the target cells in a similar manner.

Maintenance of Self-Renewal or Pluripotency

We disclose a method of maintaining or enhancing self-renewal orpluripotency, or both, in or of a stem cell. Such a method can comprisemanipulating a stem cell by a method set out above.

We therefore describe a method of manipulating a stem cell bymodulating, such as up-regulating, any combination of the expression,amount or activity of an ELABELA polypeptide in or of the stem cell. Themethod can comprise exposing the stem cell to an ELABELA polypeptide.

Alternatively, or in addition, ELABELA expression, amount or activitycan be up-regulated in the stem cell by introducing an ELABELAexpression construct (also known as an ELABELA construct) into the cell.The ELABELA expression construct or ELABELA construct can comprise anELABELA nucleic acid or a vector (such as an expression vector)comprising an ELABELA nucleic acid sequence. Methods of introducing suchconstructs are set out in detail in the immediately preceding section.

Maintenance of Pluripotency

Cells treated by the methods and compositions described here retainpluripotency. In other words, such cells retain at least onecharacteristic of a stem cell, such as a vertebrate, mammalian, primateor human stem cell. Such cells can retain the characteristic after oneor more passages. They can do so after a plurality of passages.

The pluripotency or stem cell characteristic can comprise amorphological characteristic, immunohistochemical characteristic, amolecular biological characteristic, etc. The characteristic cancomprise a biological activity.

Stem Cell Characteristics

The cells treated by our methods, in which pluripotency is retained, candisplay any of the following stem cell characteristics.

Stem cells can display increased expression of Oct4/POU5F1, TRA-1-160and/or SSEA-1. Expression of any one or more of Flk-1, Tie-2 and c-kitcan be decreased. Stem cells which are self-renewing can display ashortened cell cycle compared to stem cells which are not self-renewing.

Stem cells can display defined morphology. For example, in the twodimensions of a standard microscopic image, human embryonic stem cellsdisplay high nuclear/cytoplasmic ratios in the plane of the image,prominent nucleoli, and compact colony formation with poorly discernablecell junctions.

Stem cells can also be characterized by expressed cell markers asdescribed in further detail below.

Expression of Pluripotency Markers

The biological activity that is retained can comprise expression of apluripotency marker.

Stage-specific embryonic antigens (SSEA) are characteristic of certainembryonic cell types. Antibodies for SSEA markers are available from theDevelopmental Studies Hybridoma Bank (Bethesda Md.). Other usefulmarkers are detectable using antibodies designated Tra-1-60 and Tra-1-81(Andrews et al., Cell Linesfrom Human Gern Cell Tumors, in E. J.Robertson, 1987, supra). Human embryonic stem cells are typically SSEA-1negative and SSEA-4 positive. hEG cells are typically SSEA-1 positive.Differentiation of pPS cells in vitro results in the loss of SSEA-4,TRA-1-60, and TRA-1-81 expression and increased expression of SSEA-1.pPS cells can also be characterized by the presence of alkalinephosphatase activity, which can be detected by fixing the cells with 4%paraformaldehyde, and then developing with Vector Red as a substrate, asdescribed by the manufacturer (Vector Laboratories, Burlingame Calif.).

Embryonic stem cells are also typically telomerase positive andOCT-4/POU5F1 positive. Telomerase activity can be determined using TRAPactivity assay (Kim et al., Science 266:2011, 1997), using acommercially available kit (TRAPeze® XK Telomerase Detection Kit, Cat.s7707; Intergen Co., Purchase N.Y.; or TeloTAGGG™ Telomerase PCR ELISAplus, Cat. 2,013,89; Roche Diagnostics, Indianapolis). hTERT expressioncan also be evaluated at the mRNA level by RT-PCR. The LightCyclerTeloTAGGG™ hTERT quantification kit (Cat. 3,012,344; Roche Diagnostics)is available commercially for research purposes.

Any one or more of these pluripotency markers, including FOXD3, PODXL,alkaline phosphatase, POU5F1 (also known as OCT-4), SSEA-3, SSEA-4 andTRA-1-60, etc, can be retained by the cells produced by the methods andcompositions described here.

Detection of markers can be achieved through any means known in the art,for example immunologically. Histochemical staining, flow cytometry(FACs), Western Blot, enzyme-linked immunoassay (ELISA), etc can beused.

Flow immunocytochemistry can be used to detect cell-surface markers.immunohistochemistry (for example, of fixed cells or tissue sections)can be used for intracellular or cell-surface markers. Western blotanalysis can be conducted on cellular extracts. Enzyme-linkedimmunoassay can be used for cellular extracts or products secreted intothe medium.

For this purpose, antibodies to the pluripotency markers as availablefrom commercial sources can be used.

Antibodies for the identification of stem cell markers including theStage-Specific Embryonic Antigens 1 and 4 (SSEA-1 and SSEA-4) and TumorRejection Antigen 1-60 and 1-81 (TRA-1-60, TRA-1-81) can be obtainedcommercially, for example from Chemicon International, Inc (Temecula,Calif., USA). The immunological detection of these antigens usingmonoclonal antibodies has been widely used to characterize pluripotentstem cells (Shamblott M. J. et. al. (1998) PNAS 95: 13726-13731;Schuldiner M. et. al. (2000). PNAS 97: 11307-11312; Thomson J. A. et.al. (1998). Science 282: 1145-1147; Reubinoff B. E. et. al. (2000).Nature Biotechnology 18: 399-404; Henderson J. K. et. al. (2002). StemCells 20: 329-337; Pera M. et. al. (2000). J. Cell Science 113: 5-10.).

The expression of tissue-specific gene products can also be detected atthe mRNA level by Northern blot analysis, dot-blot hybridizationanalysis, or by reverse transcriptase initiated polymerase chainreaction (RT-PCR) using sequence-specific primers in standardamplification methods. Sequence data for the particular markers listedin this disclosure can be obtained from public databases such as GenBank(URL www.ncbi.nlm.nih.gov:80/entrez). See U.S. Pat. No. 5,843,780 forfurther details.

Substantially all of the cells treated by the methods and compositionsdescribed here, or a substantial portion of them, can express themarker(s). For example, the percentage of cells that express the markeror markers can be 50% or more, 60% or more, 70% or more, 80% or more,90% or more, 93% or more, 95% or more, 97% or more, 97% or more, 99% ormore, or substantially 100%.

Cell Viability

The biological activity can comprise cell viability after treatment bythe methods and compositions described here, or after propagationfollowing treatment. Cell viability can be assayed in various ways, forexample by Trypan Blue exclusion.

A protocol for vital staining follows. Place a suitable volume of a cellsuspension (20-200 μL) in appropriate tube add an equal volume of 0.4%Trypan blue and gently mix, let stand for 5 minutes at room temperature.Place 10 μl of stained cells in a hemocytometer and count the number ofviable (unstained) and dead (stained) cells. Calculate the averagenumber of unstained cells in each quadrant, and multiply by 2×10⁴ tofind cells/ml. The percentage of viable cells is the number of viablecells divided by the number of dead and viable cells.

The viability of cells can be 50% or more, 60% or more, 70% or more, 80%or more, 90% or more, 93% or more, 95% or more, 97% or more, 97% ormore, 99% or more, or substantially 100%.

Karyotype

The cells treated by the methods and compositions described here, inwhich pluripotency is enhanced or induced, can retain a normal karyotypeduring or after propagation. A “normal” karyotype is a karyotype that isidentical, similar or substantially similar to a karyotype of a parentstem cell from which the propagule is derived, or one which varies fromit but not in any substantial manner For example, there should not beany gross anomalies such as translocations, loss of chromosomes,deletions, etc.

Karyotype can be assessed by a number of methods, for example visuallyunder optical microscopy. Karyotypes can be prepared and analyzed asdescribed in McWhir et al. (2006), Hewitt et al. (2007), and Gallimoreand Richardson (1973). Cells can also be karyotyped using a standardG-banding technique (available at many clinical diagnostics labs thatprovides routine karyotyping services, such as the Cytogenetics Lab atOakland Calif.) and compared to published stem cell karyotypes.

All or a substantial portion of cells treated by the methods andcompositions described here can retain a normal karyotype. Thisproportion can be 50% or more, 60% or more, 70% or more, 80% or more,90% or more, 93% or more, 95% or more, 97% or more, 97% or more, 99% ormore, or substantially 100%.

Pluripotency

The cells treated by our methods can retain the capacity todifferentiate into all three embryonic lineages, i.e., endoderm,ectoderm and mesoderm. Methods of induction of stem cells todifferentiate each of these lineages are known in the art and can beused to assay the capability of the cells to differentiate. All or asubstantial portion of the treated cells can retain this ability. Thiscan be 50% or more, 60% or more, 70% or more, 80% or more, 90% or more,93% or more, 95% or more, 97% or more, 97% or more, 99% or more, orsubstantially 100% of the treated cells.

Co-Culture and Feeders

Our methods can comprise culturing stem cells in the presence or absenceof co-culture. The term “co-culture” refers to a mixture of two or moredifferent kinds of cells that are grown together, for example, stromalfeeder cells. The two or more different kinds of cells can be grown onthe same surfaces, such as particles or cell container surfaces, or ondifferent surfaces. The different kinds of cells can be grown ondifferent particles or container surfaces.

Feeder cells, as the term is used in this document, can mean cells whichare used for or required for cultivation of cells of a different type.In the context of stem cell culture, feeder cells have the function ofsecuring the survival, proliferation, and maintenance of ES-cellpluripotency. ES-cell pluripotency can be achieved by directlyco-cultivating the feeder cells. Alternatively, or in addition, thefeeder cells can be cultured in a medium to condition it. Theconditioned medium can be used to culture the stem cells.

The inner surface of the container such as a culture dish can be coatedwith a feeder layer of mouse embryonic skin cells that have been treatedso they will not divide. The feeder cells release nutrients into theculture medium which are required for ES cell growth. The stem cells canbe grown in such coated containers.

The feeder cells can themselves be grown on particles. They can beseeded on particles in a similar way as described for stem cells. Thestem cells to be propagated can be grown together with or separate fromsuch feeder particles. The stem cells can therefore be grown on a layeron such feeder cell coated particles. On the other hand, the stem cellscan be grown on separate particles. Any combinations of any of thesearrangements are also possible, for example, a culture which comprisesfeeder cells grown on particles, particles with feeder cells and stemcells, and particles with stem cells growing. These combinations can begrown in containers with a feeder layer or without.

Arrangements in which feeder cells are absent or not required are alsopossible. For example, the cells can be grown in medium conditioned byfeeder cells or stem cells.

Media and Feeder Cells

Media for isolating and propagating pluripotent stem cells can have anyof several different formulas, as long as the cells obtained have thedesired characteristics, and can be propagated further.

Suitable sources are as follows: Dulbecco's modified Eagles medium(DMEM), Gibco#11965-092; Knockout Dulbecco's modified Eagles medium (KODMEM), Gibco#10829-018; 200 mM L-glutamine, Gibco#15039-027;non-essential amino acid solution, Gibco 11140-050;beta-mercaptoethanol, Sigma#M7522; human recombinant basic fibroblastgrowth factor (bFGF), Gibco#13256-029. Exemplary serum-containingembryonic stem (ES) medium is made with 80% DMEM (typically KO DMEM),20% defined fetal bovine serum (FBS) not heat inactivated, 0.1 mMnon-essential amino acids, 1 mM L-glutamine, and 0.1 mMbeta-mercaptoethanol. The medium is filtered and stored at 4 degrees C.for no longer than 2 weeks. Serum-free embryonic stem (ES) medium ismade with 80% KO DMEM, 20% serum replacement, 0.1 mM non-essential aminoacids, 1 mM L-glutamine, and 0.1 mM beta-mercaptoethanol. An effectiveserum replacement is Gibco#10828-028. The medium is filtered and storedat 4 degrees C. for no longer than 2 weeks. Just before use, human bFGFis added to a final concentration of 4 ng/mL (Bodnar et al., Geron Corp,International Patent Publication WO 99/20741).

The media can comprise Knockout DMEM media (Invitrogen-Gibco, GrandIsland, N.Y.), supplemented with 10% serum replacement media(Invitrogen-Gibco, Grand Island, N.Y.), 5ng/ml FGF2 (Invitrogen-Gibco,Grand Island, N.Y.) and 5ng/ml PDGF AB (Peprotech, Rocky Hill, N.J.).

Feeder cells (where used) can be propagated in mEF medium, containing90% DMEM (Gibco#11965-092), 10% FBS (Hyclone#30071-03), and 2 mMglutamine. mEFs are propagated in T150 flasks (Coming#430825), splittingthe cells 1:2 every other day with trypsin, keeping the cellssubconfluent. To prepare the feeder cell layer, cells are irradiated ata dose to inhibit proliferation but permit synthesis of importantfactors that support human embryonic stem cells (.about.4000 rads gammairradiation). Six-well culture plates (such as Falcon#304) are coated byincubation at 37 degrees C. with 1 mL 0.5% gelatin per well overnight,and plated with 375,000 irradiated mEFs per well. Feeder cell layers aretypically used 5 h to 4 days after plating. The medium is replaced withfresh human embryonic stem (hES) medium just before seeding pPS cells.

Conditions for culturing other stem cells are known, and can beoptimized appropriately according to the cell type. Media and culturetechniques for particular cell types referred to in the previous sectionare provided in the references cited.

Serum Free Media

The methods and compositions described here can include culture of cellssuch as stem cells in a serum-free medium.

The term “serum-free media” can comprise cell culture media which isfree of serum proteins, e.g., fetal calf serum. Serum-free media areknown in the art, and are described for example in U.S. Pat. Nos.5,631,159 and 5,661,034. Serum-free media are commercially availablefrom, for example, Gibco-BRL (Invitrogen).

The serum-free media can be protein free, in that it can lack proteins,hydrolysates, and components of unknown composition. The serum-freemedia can comprise chemically-defined media in which all components havea known chemical structure. Chemically-defined serum-free media isadvantageous as it provides a completely defined system which eliminatesvariability allows for improved reproducibility and more consistentperformance, and decreases possibility of contamination by adventitiousagents.

The serum-free media can comprise Knockout DMEM media (Invitrogen-Gibco,Grand Island, N.Y.).

The serum-free media can be supplemented with one or more components,such as serum replacement media, at a concentration of for example, 5%,10%, 15%, etc. The serum-free media can be supplemented with 10% serumreplacement media from Invitrogen-Gibco (Grand Island, N.Y.).

The serum-free medium in which the dissociated or disaggregatedembryonic stem cells are cultured can comprise one or more growthfactors. A number of growth factors are known in the art, includingFGF2, IGF-2, Noggin, Activin A, TGF beta 1, HRG1 beta, LIF, S1P, PDGF,BAFF, April, SCF, Flt-3 ligand, Wnt3A and others. The growth factor(s),can be used at any suitable concentration such as between 1 pg/ml to 500ng/ml.

Stem Cells

As used in this document, the term “stem cell” refers to a cell that ondivision faces two developmental options: the daughter cells can beidentical to the original cell (self-renewal) or they can be theprogenitors of more specialised cell types (differentiation). The stemcell is therefore capable of adopting one or other pathway (a furtherpathway exists in which one of each cell type can be formed). Stem cellsare therefore cells which are not terminally differentiated and are ableto produce cells of other types.

Stem cells as referred to in this document can include totipotent stemcells, pluripotent stem cells, and multipotent stem cells. They alsospecifically include induced pluripotent stem cells (iPS).

Totipotent Stem Cells

The term “totipotent” cell refers to a cell which has the potential tobecome any cell type in the adult body, or any cell of theextraembryonic membranes (e.g., placenta). Thus, the only totipotentcells are the fertilized egg and the first 4 or so cells produced by itscleavage.

Pluripotent Stem Cells

“Pluripotent stem cells” are true stem cells, with the potential to makeany differentiated cell in the body. However, they cannot contribute tomaking the extraembryonic membranes which are derived from thetrophoblast. Several types of pluripotent stem cells have been found.

Embryonic Stem Cells

Embryonic Stem (ES) cells can be isolated from the inner cell mass (ICM)of the blastocyst, which is the stage of embryonic development whenimplantation occurs.

Embryonic Germ Cells

Embryonic Germ (EG) cells can be isolated from the precursor to thegonads in aborted fetuses.

Embryonic Carcinoma Cells

Embryonic Carcinoma (EC) cells can be isolated from teratocarcinomas, atumor that occasionally occurs in a gonad of a fetus. Unlike the firsttwo, they are usually aneuploid. All three of these types of pluripotentstem cells can only be isolated from embryonic or fetal tissue and canbe grown in culture. Methods are known in the art which prevent thesepluripotent cells from differentiating.

Adult Stem Cells

Adult stem cells comprise a wide variety of types including neuronal,skin and the blood forming stem cells which are the active component inbone marrow transplantation. These latter stem cell types are also theprincipal feature of umbilical cord-derived stem cells. Adult stem cellscan mature both in the laboratory and in the body into functional, morespecialised cell types although the exact number of cell types islimited by the type of stem cell chosen.

Multipotent Stem Cells

Multipotent stem cells are true stem cells but can only differentiateinto a limited number of types. For example, the bone marrow containsmultipotent stem cells that give rise to all the cells of the blood butnot to other types of cells. Multipotent stem cells are found in adultanimals. It is thought that every organ in the body (brain, liver)contains them where they can replace dead or damaged cells.

Methods of characterising stem cells are known in the art, and includethe use of standard assay methods such as clonal assay, flow cytometry,long-term culture and molecular biological techniques e.g. PCR, RT-PCRand Southern blotting.

In addition to morphological differences, human and murine pluripotentstem cells differ in their expression of a number of cell surfaceantigens (stem cell markers). Markers for stem cells and methods oftheir detection are described elsewhere in this document (under“Maintenance of Stem Cell Characteristics”).

Screening for Anti-Elabela Agents Identifying ELABELA Modulators,Agonists and Antagonists

Antagonists, in particular, small molecules can be used to specificallyinhibit ELABELA for use as anti-ELABELA agents.

We disclose a method of assaying a compound of interest, the methodcomprising contacting an ELABELA polypeptide with a candidate compoundand performing an assay to determine if the candidate compound binds tothe ELABELA polypeptide.

We further disclose a method of assaying a compound of interest, themethod comprising contacting an ELABELA polypeptide with a candidatecompound and performing an assay to determine if the candidate compoundmodulates an activity of the ELABELA polypeptide.

We further disclose a method of assaying a compound of interest, themethod comprising contacting a cell expressing an ELABELA polypeptidewith a candidate compound and performing an assay to determine if thecandidate compound causes an elevated or reduced expression, amount oractivity of the ELABELA polypeptide in or of the cell.

The compound of interest so identified can then be isolated orchemically synthesised.

We therefore disclose ELABELA antagonists and small molecule ELABELAinhibitors, as well as assays for screening for these. Antagonists ofELABELA can be screened by detecting modulation, such as downregulation, of binding or other ELABELA activity. We therefore provide acompound capable of down-regulating the expression, amount or activityELABELA polypeptide. Such a compound can be used in the methods andcompositions described here for treating or preventing an ELABELAassociated condition.

ELABELA can therefore be used to assess the binding of small moleculesubstrates and ligands in, for example, cells, cell-free preparations,chemical libraries, and natural product mixtures. These substrates andligands can be natural substrates and ligands or can be structural orfunctional mimetics. See Coligan et al., Current Protocols in Immunology1(2):Chapter 5 (1991). Furthermore, screens can be conducted to identifyfactors which influence the expression of ELABELA, in particular in anELABELA associated condition.

In general, the assays for agonists and antagonists rely on determiningthe effect of candidate molecules on one or more activities of ELABELA.An assay can involve assaying ELABELA activity in the presence of acandidate molecule, and optionally in the absence of the candidatemolecule, or in the presence of a molecule known to inhibit or activatea ELABELA activity.

Expression of ELABELA can be modulated, such as up-regulated in anELABELA associated condition. Therefore, it can be desirous to findcompounds and drugs which stimulate the expression and/or activity ofELABELA, or which can inhibit the function of this protein. In general,agonists and antagonists can be employed for therapeutic andprophylactic purposes for an ELABELA associated condition.

By “down-regulation” we include any negative effect on the behaviourbeing studied; this can be total or partial. Thus, where binding isbeing detected, candidate antagonists are capable of reducing,ameliorating, or abolishing the binding between two entities. Thedown-regulation of binding (or any other activity) achieved by thecandidate molecule can be at least 10%, such as at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90% or more compared to binding (or which ever activity)in the absence of the candidate molecule. Thus, a candidate moleculesuitable for use as an antagonist is one which is capable of reducing by10% more the binding or other activity.

The term “compound” refers to a chemical compound (naturally occurringor synthesised), such as a biological macromolecule (e.g., nucleic acid,protein, non-peptide, or organic molecule), or an extract made frombiological materials such as bacteria, plants, fungi, or animal(particularly mammalian) cells or tissues, or even an inorganic elementor molecule. The compound can be an antibody.

Examples of potential antagonists of ELABELA include antibodies, smallmolecules, nucleotides and their analogues, including purines and purineanalogues, oligonucleotides or proteins which are closely related to abinding partner of ELABELA, e.g., a fragment of the binding partner, orsmall molecules which bind to the ELABELA polypeptide but do not elicita response, so that the activity of the polypeptide is prevented, etc.

Screening Kits

The materials necessary for such screening to be conducted can bepackaged into a screening kit.

Such a screening kit is useful for identifying agonists, antagonists,ligands, receptors, substrates, enzymes, etc. for ELABELA polypeptidesor compounds which decrease or enhance the production of ELABELA. Thescreening kit can comprise: (a) a ELABELA polypeptide; (b) a recombinantcell expressing a ELABELA polypeptide; or (c) an antibody to ELABELApolypeptide. The screening kit can comprise a library. The screening kitcan comprise any one or more of the components needed for screening, asdescribed elsewhere in this document. The screening kit can optionallycomprise instructions for use.

Screening kits can also be provided which are capable of detectingELABELA expression at the nucleic acid level. Such kits can comprise aprimer for amplification of ELABELA, or a pair of primers foramplification. The primer or primers can be chosen from any suitablesequence, for example a portion of the ELABELA sequence. Methods ofidentifying primer sequences are well known in the art, and the skilledperson will be able to design such primers with ease. The kits cancomprise a nucleic acid probe for ELABELA expression, as described inthis document. The kits can also optionally comprise instructions foruse.

Rational Design

Rational design of candidate compounds likely to be able to interactwith ELABELA can be based upon structural studies of the molecularshapes of a ELABELA polypeptide. One means for determining which sitesinteract with specific other proteins is a physical structuredetermination, e.g., X-ray crystallography or two-dimensional NMRtechniques. These will provide guidance as to which amino acid residuesform molecular contact regions. For a detailed description of proteinstructural determination, see, e.g., Blundell and Johnson (1976) ProteinCrystallography, Academic Press, New York.

Polypeptide Binding Assays

Modulators and antagonists of ELABELA activity or expression can beidentified by any means known in the art.

In their simplest form, the assays can simply comprise the steps ofmixing a candidate compound with a solution containing a ELABELApolypeptide to form a mixture, measuring activity of ELABELA polypeptidein the mixture, and comparing the activity of the mixture to a standard.

Furthermore, molecules can be identified by their binding to ELABELA, inan assay which detects binding between ELABELA and the putativemolecule.

One type of assay for identifying substances that bind to a ELABELApolypeptide described here involves contacting the ELABELA polypeptide,which is immobilised on a solid support, with a non-immobilisedcandidate substance determining whether and/or to what extent theELABELA polypeptide of interest and candidate substance bind to eachother. Alternatively, the candidate substance can be immobilised and theELABELA polypeptide as set out in this document non-immobilised.

The binding of the substance to the ELABELA polypeptide can betransient, reversible or permanent. The substance can bind to thepolypeptide with a Kd value which is lower than the Kd value for bindingto control polypeptides (e.g., polypeptides known to not be involved inan ELABELA associated condition). The Kd value of the substance can be 2fold less than the Kd value for binding to control polypeptides, such asa Kd value 100 fold less or a Kd 1000 fold less than that for binding tothe control polypeptide.

In an example assay method, the ELABELA polypeptide can be immobilisedon beads such as agarose beads. Typically this can be achieved byexpressing the ELABELA polypeptide as a GST-fusion protein in bacteria,yeast or higher eukaryotic cell lines and purifying the GST-ELABELAfusion protein from crude cell extracts using glutathione-agarose beads(Smith and Johnson, 1988; Gene 67(10):31-40). As a control, binding ofthe candidate substance, which is not a GST-fusion protein, to animmobilised polypeptide can be determined in the absence of the ELABELApolypeptide. The binding of the candidate substance to the immobilisedELABELA polypeptide can then be determined. This type of assay is knownin the art as a GST pulldown assay. Again, the candidate substance canbe immobilised and the ELABELA polypeptide non-immobilised.

It is also possible to perform this type of assay using differentaffinity purification systems for immobilising one of the components,for example Ni-NTA agarose, histidine-tagged components as well asantibody-based affinity chromatography.

Binding of the polypeptide to the candidate substance can be determinedby a variety of methods well-known in the art. For example, thenon-immobilised component can be labeled (with for example, aradioactive label, an epitope tag or an enzyme-antibody conjugate).Alternatively, binding can be determined by immunological detectiontechniques. For example, the reaction mixture can be Western blotted andthe blot probed with an antibody that detects the non-immobilisedcomponent. ELISA techniques can also be used.

Candidate substances are typically added to a final concentration offrom 1 to 1000 nmol/ml, such as from 1 to 100 nmol/ml. In the case ofantibodies, the final concentration used is typically from 100 to 500μg/ml, such as from 200 to 300 μg/ml.

Modulators and antagonists of ELABELA can also be identified bydetecting modulation of binding between ELABELA and any molecule towhich this polypeptide binds, or modulation of any activityconsequential on such binding or release.

Cell Based Assays

A cell based assay can simply test binding of a candidate compoundwherein adherence to the cells bearing the ELABELA polypeptide isdetected by means of a label directly or indirectly associated with thecandidate compound or in an assay involving competition with a labelledcompetitor.

Further, these assays can test whether the candidate compound results ina signal generated by binding to the ELABELA polypeptide, usingdetection systems appropriate to the cells bearing the polypeptides attheir surfaces Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed.

Another method of screening compounds utilises eukaryotic or prokaryotichost cells which are stably transformed with recombinant DNA moleculesexpressing a library of compounds. Such cells, either in viable or fixedform, can be used for standard binding-partner assays. See also Parce etal. (1989) Science 246:243-247; and Owicki et al. (1990) Proc. Nat'lAcad. Sci. USA 87;4007-4011, which describe sensitive methods to detectcellular responses.

Competitive assays are particularly useful, where the cells expressingthe library of compounds are contacted or incubated with a labelledantibody known to bind to a ELABELA polypeptide, such as ¹²⁵I-antibody,and a test sample such as a candidate compound whose binding affinity tothe binding composition is being measured. The bound and free labelledbinding partners for the ELABELA polypeptide are then separated toassess the degree of binding. The amount of test sample bound isinversely proportional to the amount of labelled antibody binding to theELABELA polypeptide.

Any one of numerous techniques can be used to separate bound from freebinding partners to assess the degree of binding. This separation stepcould typically involve a procedure such as adhesion to filters followedby washing, adhesion to plastic following by washing, or centrifugationof the cell membranes.

The assays can involve exposing a candidate molecule to a cell, such asa colon, lung, squamous cell including lip, larynx, vulva, cervix andpenis, pancreatic, brain, oesophageal, stomach, bladder, kidney, skin,ovary, prostate and testicular cell, and assaying expression of ELABELAby any suitable means. Molecules which down-regulate the expression ofELABELA in such assays can be optionally chosen for further study, andused as drugs to down-regulate ELABELA expression. Such drugs can beusefully employed to treat or prevent an ELABELA associated condition.

cDNA encoding ELABELA protein and antibodies to the proteins can also beused to configure assays for detecting the effect of added compounds onthe production of ELABELA mRNA and protein in cells. For example, anELISA can be constructed for measuring secreted or cell associatedlevels of ELABELA polypeptide using monoclonal and polyclonal antibodiesby standard methods known in the art, and this can be used to discoveragents which can inhibit or enhance the production of ELABELA protein(also called antagonist or agonist, respectively) from suitablymanipulated cells or tissues. Standard methods for conducting screeningassays are well understood in the art.

Activity Assays

Assays to detect modulators or antagonists typically involve detectingmodulation of any activity of ELABELA, in the presence, optionallytogether with detection of modulation of activity in the absence, of acandidate molecule.

Assays which detect specific biological activities of ELABELA, such asphosphatase activity, can be used. The assays typically involvecontacting a candidate molecule (e.g., in the form of a library) withELABELA whether in the form of a polypeptide, a nucleic acid encodingthe polypeptide, or a cell, organelle, extract, or other materialcomprising such, with a candidate modulator. The relevant activity ofELABELA (such as phosphatase activity, as described elsewhere in thisdocument) can be detected, to establish whether the presence of thecandidate modulator has any effect.

Phosphatase assays are known in the art and are described in Wu et al(2004), Int J Biochem Cell Biol. 36(8):1542-53 and Alonso et al (2004).J Biol Chem. 20;279(34):35768-74. Such assays comprise assaying theability of ELABELA to de-phosphorylate a suitable substrate such asp-nitrophenyl phosphate, or as oligopeptides containing phospho-tyrosineand phospho-threonine residues. The assays can be performed in thepresence or absence of a candidate modulator and the appropriateactivity detected to detect modulation of ELABELA activity and henceidentification of a candidate modulator and/or antagonist of ELABELA.

Promoter binding assays to detect candidate modulators which bind toand/or affect the transcription or expression of ELABELA can also beused. Candidate modulators can then be chosen for further study, orisolated for use. Details of such screening procedures are well known inthe art, and are for example described in, Handbook of Drug Screening,edited by Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, NewYork, N.Y., Marcel Dekker, ISBN 0-8247-0562-9).

The screening methods described here can employ in vivo assays, althoughthey can be configured for in vitro use. In vivo assays generallyinvolve exposing a cell comprising ELABELA to the candidate molecule. Inin vitro assays, ELABELA is exposed to the candidate molecule,optionally in the presence of other components, such as crude orsemi-purified cell extract, or purified proteins. Where in vitro assaysare conducted, these can employ arrays of candidate molecules (forexample, an arrayed library). In vivo assays can be employed. Therefore,the ELABELA polypeptide can be comprised in a cell, such asheterologously. Such a cell can be a transgenic cell, which has beenengineered to express ELABELA as described above.

Where an extract is employed, it can comprise a cytoplasmic extract or anuclear extract, methods of preparation of which are well known in theart.

It will be appreciated that any component of a cell comprising ELABELAcan be employed, such as an organelle. One embodiment utilises acytoplasmic or nuclear preparation, e.g., comprising a cell nucleuswhich comprises ELABELA as described. The nuclear preparation cancomprise one or more nuclei, which can be permeabilised orsemi-permeabilised, by detergent treatment, for example.

Thus, in a specific embodiment, an assay format can include thefollowing: a multiwell microtitre plate is set up to include one or morecells expressing ELABELA polypeptide in each well; individual candidatemolecules, or pools of candidate molecules, derived for example from alibrary, can be added to individual wells and modulation of ELABELAactivity measured. Where pools are used, these can be subdivided in tofurther pools and tested in the same manner ELABELA activity, forexample binding activity or transcriptional co-activation activity, asdescribed elsewhere in this document can then be assayed.

Alternatively or in addition to the assay methods described above,“subtractive” procedures can also be used to identify modulators orantagonists of ELABELA. Under such “subtractive” procedures, a pluralityof molecules is provided, which comprises one or more candidatemolecules capable of functioning as a modulator (e.g., cell extract,nuclear extract, library of molecules, etc), and one or more componentsis removed, depleted or subtracted from the plurality of molecules. The“subtracted” extract, etc, is then assayed for activity, by exposure toa cell comprising ELABELA (or a component thereof) as described.

Thus, for example, an ‘immunodepletion’ assay can be conducted toidentify such modulators as follows. A cytoplasmic or nuclear extractcan be prepared from a suitable cell. The extract can be depleted orfractionated to remove putative modulators, such as by use ofimmunodepletion with appropriate antibodies. If the extract is depletedof a modulator, it will lose the ability to affect ELABELA function oractivity or expression. A series of subtractions and/or depletions canbe required to identify the modulators or antagonists.

It will also be appreciated that the above “depletion” or “subtraction”assay can be used as a preliminary step to identify putative modulatoryfactors for further screening. Furthermore, or alternatively, the“depletion” or “subtraction” assay can be used to confirm the modulatoryactivity of a molecule identified by other means (for example, a“positive” screen as described elsewhere in this document) as a putativemodulator.

Candidate molecules subjected to the assay and which are found to be ofinterest can be isolated and further studied. Methods of isolation ofmolecules of interest will depend on the type of molecule employed,whether it is in the form of a library, how many candidate molecules arebeing tested at any one time, whether a batch procedure is beingfollowed, etc.

The candidate molecules can be provided in the form of a library. In oneembodiment, more than one candidate molecule can be screenedsimultaneously. A library of candidate molecules can be generated, forexample, a small molecule library, a polypeptide library, a nucleic acidlibrary, a library of compounds (such as a combinatorial library), alibrary of antisense molecules such as antisense DNA or antisense RNA,an antibody library etc, by means known in the art. Such libraries aresuitable for high-throughput screening. Different cells comprisingELABELA can be exposed to individual members of the library, and effecton the ELABELA activity determined. Array technology can be employed forthis purpose. The cells can be spatially separated, for example, inwells of a microtitre plate.

In an embodiment, a small molecule library is employed. By a “smallmolecule”, we refer to a molecule whose molecular weight can be lessthan about 50 kDa. In particular embodiments, a small molecule can havea molecular weight which is less than about 30 kDa, such as less thanabout 15 kDa or less than 10 kDa or so. Libraries of such smallmolecules, here referred to as “small molecule libraries” can containpolypeptides, small peptides, for example, peptides of 20 amino acids orfewer, for example, 15, 10 or 5 amino acids, simple compounds, etc.

Alternatively or in addition, a combinatorial library, as described infurther detail elsewhere in this document, can be screened formodulators or antagonists of ELABELA. Assays for ELABELA activity aredescribed above.

Libraries

Libraries of candidate molecules, such as libraries of polypeptides ornucleic acids, can be employed in the screens for ELABELA antagonistsand inhibitors described here. Such libraries are exposed to ELABELAprotein, and their effect, if any, on the activity of the proteindetermined.

Selection protocols for isolating desired members of large libraries areknown in the art, as typified by phage display techniques. Such systems,in which diverse peptide sequences are displayed on the surface offilamentous bacteriophage (Scott and Smith (1990 supra), have provenuseful for creating libraries of antibody fragments (and the nucleotidesequences that encoding them) for the in vitro selection andamplification of specific antibody fragments that bind a target antigen.The nucleotide sequences encoding the V_(H) and V_(L) regions are linkedto gene fragments which encode leader signals that direct them to theperiplasmic space of E. coli and as a result the resultant antibodyfragments are displayed on the surface of the bacteriophage, typicallyas fusions to bacteriophage coat proteins (e.g., pIII or pVIII).Alternatively, antibody fragments are displayed externally on lambdaphage capsids (phagebodies). An advantage of phage-based display systemsis that, because they are biological systems, selected library memberscan be amplified simply by growing the phage containing the selectedlibrary member in bacterial cells. Furthermore, since the nucleotidesequence that encodes the polypeptide library member is contained on aphage or phagemid vector, sequencing, expression and subsequent geneticmanipulation is relatively straightforward.

Methods for the construction of bacteriophage antibody display librariesand lambda phage expression libraries are well known in the art(McCafferty et al. (1990) supra; Kang et al. (1991) Proc. Natl. Acad.Sci. U.S.A., 88: 4363; Clackson et al. (1991) Nature, 352: 624; Lowmanet al. (1991) Biochemistry, 30: 10832; Burton et al. (1991) Proc. Natl.Acad. Sci U.S.A., 88: 10134; Hoogenboom et al. (1991) Nucleic AcidsRes., 19: 4133; Chang et al. (1991) J. Immunol., 147: 3610; Breitling etal. (1991) Gene, 104: 147; Marks et al. (1991) supra; Barbas et al.(1992) supra; Hawkins and Winter (1992) J. Immunol., 22: 867; Marks etal., 1992, J. Biol. Chem., 267: 16007; Lerner et al. (1992) Science,258: 1313, incorporated herein by reference). Such techniques can bemodified if necessary for the expression generally of polypeptidelibraries.

One particularly advantageous approach has been the use of scFvphage-libraries (Bird, R. E., et al. (1988) Science 242: 423-6, Hustonet al., 1988, Proc. Natl. Acad. Sci U.S.A., 85: 5879-5883; Chaudhary etal. (1990) Proc. Natl. Acad. Sci U.S.A., 87: 1066-1070; McCafferty etal. (1990) supra; Clackson et al. (1991) supra; Marks et al. (1991)supra; Chiswell et al. (1992) Trends Biotech., 10: 80; Marks et al.(1992) supra). Various embodiments of scFv libraries displayed onbacteriophage coat proteins have been described. Refinements of phagedisplay approaches are also known, for example as described inWO96/06213 and WO92/01047 (Medical Research Council et al.) andWO97/08320 (Morphosys, supra), which are incorporated herein byreference.

Alternative library selection technologies include bacteriophage lambdaexpression systems, which can be screened directly as bacteriophageplaques or as colonies of lysogens, both as previously described (Huseet al. (1989) Science, 246: 1275; Caton and Koprowski (1990) Proc. Natl.Acad. Sci. U.S.A., 87; Mullinax et al. (1990) Proc. Natl. Acad. Sci.U.S.A., 87: 8095; Persson et al. (1991) Proc. Natl. Acad. Sci. U.S.A.,88: 2432) and are of use in the methods and compositions described here.These expression systems can be used to screen a large number ofdifferent members of a library, in the order of about 10⁶ or even more.Other screening systems rely, for example, on direct chemical synthesisof library members. One early method involves the synthesis of peptideson a set of pins or rods, such as described in WO84/03564. A similarmethod involving peptide synthesis on beads, which forms a peptidelibrary in which each bead is an individual library member, is describedin U.S. Pat. No. 4,631,211 and a related method is described inWO92/00091. A significant improvement of the bead-based methods involvestagging each bead with a unique identifier tag, such as anoligonucleotide, so as to facilitate identification of the amino acidsequence of each library member. These improved bead-based methods aredescribed in WO93/06121.

Another chemical synthesis method involves the synthesis of arrays ofpeptides (or peptidomimetics) on a surface in a manner that places eachdistinct library member (e.g., unique peptide sequence) at a discrete,predefined location in the array. The identity of each library member isdetermined by its spatial location in the array. The locations in thearray where binding interactions between a predetermined molecule (e.g.,a receptor) and reactive library members occur is determined, therebyidentifying the sequences of the reactive library members on the basisof spatial location. These methods are described in U.S. Pat. No.5,143,854; WO90/15070 and WO92/10092; Fodor et al. (1991) Science, 251:767; Dower and Fodor (1991) Ann. Rep. Med. Chem., 26: 271.

Other systems for generating libraries of polypeptides or nucleotidesinvolve the use of cell-free enzymatic machinery for the in vitrosynthesis of the library members. In one method, RNA molecules areselected by alternate rounds of selection against a target ligand andPCR amplification (Tuerk and Gold (1990) Science, 249: 505; Ellingtonand Szostak (1990) Nature, 346: 818). A similar technique can be used toidentify DNA sequences which bind a predetermined human transcriptionfactor (Thiesen and Bach (1990) Nucleic Acids Res., 18: 3203; Beaudryand Joyce (1992) Science, 257: 635; WO92/05258 and WO92/14843). In asimilar way, in vitro translation can be used to synthesise polypeptidesas a method for generating large libraries. These methods whichgenerally comprise stabilised polysome complexes, are described furtherin WO88/08453, WO90/05785, WO90/07003, WO91/02076, WO91/05058, andWO92/02536. Alternative display systems which are not phage-based, suchas those disclosed in WO95/22625 and WO95/11922 (Affymax) use thepolysomes to display polypeptides for selection. These and all theforegoing documents also are incorporated herein by reference.

Combinatorial Libraries

Libraries, in particular, libraries of candidate molecules, can suitablybe in the form of combinatorial libraries (also known as combinatorialchemical libraries).

A “combinatorial library”, as the term is used in this document, is acollection of multiple species of chemical compounds that consist ofrandomly selected subunits. Combinatorial libraries can be screened formolecules which are capable of inhibiting ELABELA.

Various combinatorial libraries of chemical compounds are currentlyavailable, including libraries active against proteolytic andnon-proteolytic enzymes, libraries of agonists and antagonists ofG-protein coupled receptors (GPCRs), libraries active against non-GPCRtargets (e.g., integrins, ion channels, domain interactions, nuclearreceptors, and transcription factors) and libraries of whole-celloncology and anti-infective targets, among others. A comprehensivereview of combinatorial libraries, in particular their construction anduses is provided in Dolle and Nelson (1999), Journal of CombinatorialChemistry, Vol 1 No 4, 235-282. Reference is also made to Combinatorialpeptide library protocols (edited by Shmuel Cabilly, Totowa, N.J.:Humana Press, c 1998. Methods in Molecular Biology v. 87). Specificcombinatorial libraries and methods for their construction are disclosedin U.S. Pat. No. 6,168,914 (Campbell, et al), as well as in Baldwin etal. (1995), “Synthesis of a Small Molecule Library Encoded withMolecular Tags,” J. Am. Chem. Soc. 117:5588-5589, and in the referencesmentioned in those documents.

In one embodiment, the combinatorial library which is screened is onewhich is designed to potentially include molecules which interact with acomponent of the cell to influence gene expression. For example,combinatorial libraries against chromatin structural proteins can bescreened. Other libraries which are useful for this embodiment includecombinatorial libraries against histone modification enzymes (e.g.,histone acetylation or histone metylation enzymes), or DNA modification,for example, DNA methylation or demethylation.

Further references describing chemical combinatorial libraries, theirproduction and use include those available from the URLhttp://www.netsci.org/Science/Combichem/, including The ChemicalGeneration of Molecular Diversity. Michael R. Pavia, SphinxPharmaceuticals, A Division of Eli Lilly (Published July, 1995);Combinatorial Chemistry: A Strategy for the Future—MDL InformationSystems discusses the role its Project Library plays in managingdiversity libraries (Published July, 1995); Solid Support CombinatorialChemistry in Lead Discovery and SAR Optimization, Adnan M. M. Mjalli andBarry E. Toyonaga, Ontogen Corporation (Published July, 1995);Non-Peptidic Bradykinin Receptor Antagonists From a StructurallyDirected Non-Peptide Library. Sarvajit Chakravarty, Babu J. Mavunkel,Robin Andy, Donald J. Kyle*, Scios Nova Inc. (Published July, 1995);Combinatorial Chemistry Library Design using Pharmacophore DiversityKeith Davies and Clive Briant, Chemical Design Ltd. (Published July,1995); A Database System for Combinatorial Synthesis Experiments—CraigJames and David Weininger, Daylight Chemical Information Systems, Inc.(Published July, 1995); An Information Management Architecture forCombinatorial Chemistry, Keith Davies and Catherine White, ChemicalDesign Ltd. (Published July, 1995); Novel Software Tools for AddressingChemical Diversity, R. S. Pearlman, Laboratory for Molecular Graphicsand Theoretical Modeling, College of Pharmacy, University of Texas(Published June/July, 1996); Opportunities for Computational ChemistsAfforded by the New Strategies in Drug Discovery: An Opinion, YvonneConnolly Martin, Computer Assisted Molecular Design Project, AbbottLaboratories (Published June/July, 1996); Combinatorial Chemistry andMolecular Diversity Course at the University of Louisville: ADescription, Arno F. Spatola, Department of Chemistry, University ofLouisville (Published June/July, 1996); Chemically Generated ScreeningLibraries: Present and Future. Michael R. Pavia, Sphinx Pharmaceuticals,A Division of Eli Lilly (Published June/July, 1996); Chemical StrategiesFor Introducing Carbohydrate Molecular Diversity Into The Drug DiscoveryProcess. Michael J. Sofia, Transcell Technologies Inc. (PublishedJune/July, 1996); Data Management for Combinatorial Chemistry. MaryjoZaborowski, Chiron Corporation and Sheila H. DeWitt, Parke-DavisPharmaceutical Research, Division of Warner-Lambert Company (PublishedNovember, 1995); and The Impact of High Throughput Organic Synthesis onR&D in Bio-Based Industries, John P. Devlin (Published March, 1996).

Techniques in combinatorial chemistry are gaining wide acceptance amongmodern methods for the generation of new pharmaceutical leads (Gallop,M. A. et al., 1994, J. Med. Chem. 37:1233-1251; Gordon, E. M. et al.,1994, J. Med. Chem. 37:1385-1401). One combinatorial approach in use isbased on a strategy involving the synthesis of libraries containing adifferent structure on each particle of the solid phase support,interaction of the library with a soluble receptor, identification ofthe ‘bead’ which interacts with the macromolecular target, anddetermination of the structure carried by the identified ‘bead’ (Lam, K.S. et al., 1991, Nature 354:82-84). An alternative to this approach isthe sequential release of defined aliquots of the compounds from thesolid support, with subsequent determination of activity in solution,identification of the particle from which the active compound wasreleased, and elucidation of its structure by direct sequencing (Salmon,S. E. et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712), or byreading its code (Kerr, J. M. et al., 1993, J. Am. Chem. Soc.115:2529-2531; Nikolaiev, V. et al., 1993, Pept. Res. 6:161-170;Ohlmeyer, M. H. J. et al., 1993, Proc. Natl. Acad. Sci. USA90:10922-10926).

Soluble random combinatorial libraries can be synthesized using a simpleprinciple for the generation of equimolar mixtures of peptides which wasfirst described by Furka (Furka, A. et al., 1988, Xth InternationalSymposium on Medicinal Chemistry, Budapest 1988; Furka, A. et al., 1988,14th International Congress of Biochemistry, Prague 1988; Furka, A. etal., 1991, Int. J. Peptide Protein Res. 37:487-493). The construction ofsoluble libraries for iterative screening has also been described(Houghten, R. A. et al. 1991, Nature 354:84-86). K. S. Lam disclosed thenovel and unexpectedly powerful technique of using insoluble randomcombinatorial libraries. Lam synthesized random combinatorial librarieson solid phase supports, so that each support had a test compound ofuniform molecular structure, and screened the libraries without priorremoval of the test compounds from the support by solid phase bindingprotocols (Lam, K. S. et al., 1991, Nature 354:82-84).

Thus, a library of candidate molecules can be a synthetic combinatoriallibrary (e.g., a combinatorial chemical library), a cellular extract, abodily fluid (e.g., urine, blood, tears, sweat, or saliva), or othermixture of synthetic or natural products (e.g., a library of smallmolecules or a fermentation mixture).

A library of molecules can include, for example, amino acids,oligopeptides, polypeptides, proteins, or fragments of peptides orproteins; nucleic acids (e.g., antisense; DNA; RNA; or peptide nucleicacids, PNA); aptamers; or carbohydrates or polysaccharides. Each memberof the library can be singular or can be a part of a mixture (e.g., acompressed library). The library can contain purified compounds or canbe “dirty” (i.e., containing a significant quantity of impurities).

Commercially available libraries (e.g., from Affymetrix, ArQule, NeoseTechnologies, Sarco, Ciddco, Oxford Asymmetry, Canbridge, Aldrich,Panlabs, Pharmacopoeia, Sigma, or Tripose) can also be used with themethods described here.

In addition to libraries as described above, special libraries calleddiversity files can be used to assess the specificity, reliability, orreproducibility of the new methods. Diversity files contain a largenumber of compounds (e.g., 1000 or more small molecules) representativeof many classes of compounds that could potentially result innonspecific detection in an assay. Diversity files are commerciallyavailable or can also be assembled from individual compoundscommercially available from the vendors listed above.

Antibodies

Anti-ELABELA agents, including antagonists or modulators of ELABELA,which can be used to regulate the activity of this protein (for example,for methods of treating or preventing diseases such as an ELABELAassociated condition as described in this document) can includeantibodies against the ELABELA protein.

We therefore provide for antibodies which bind to a ELABELA polypeptide,fragment, homologue, variant or derivative thereof. Such antibodies canbe useful in detecting ELABELA expression, and in particular indiagnosing a ELABELA associated disease or condition. Other antibodiesinclude those which have therapeutic activity, i.e., which are can beused in a therapeutic manner to treat, manage or prevent any ELABELAassociated disease or condition.

An antibody against ELABELA can be generated by any means known in theart, from the ELABELA sequences disclosed in this document.

For example, an anti-ELABELA antibody can be generated against anELABELA polypeptide as disclosed in this document, or a polypeptideencoded by an ELABELA nucleic acid as disclosed in this document, suchas any of the ELABELA sequences set out in the sequence listing.

An antibody against ELABELA can be generated as described in theExamples, by immunisation with a peptide CMPLHSRVPFP (SEQ ID NO: 52)corresponding to amino acid residues (44-54) of human ELABELA.

Antibodies against a polypeptide comprising the sequenceMRFQQFLFAFFIFIMSLLLISG (SEQ ID NO: 19) or QRPVNLTMRRKLRKHNC (SEQ ID NO:53); or a polypeptide comprising the sequenceQRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP (SEQ ID NO: 2) can also be generated asanti-ELABELA antibodies.

Furthermore, antibodies which are specific for ELABELA can be generatedagainst any suitable epitope, for example, an epitope derived from theELABELA protein. The sequence of a suitable fragment of ELABELA cancomprise residues HSRVPFP (SEQ ID NO: 58), RCXXXHSRVPFP (SEQ ID NO: 59)or CXXXRCXXXHSRVPFP (SEQ ID NO: 1) of ELABELA and any epitope from thissequence can be used for the generation of specific ELABELA antibodies.

Accordingly, provided herein in some aspects are isolated antibodies orantigen-binding fragments thereof that specifically bind to one or moreof the following:

(a) a polypeptide comprising the sequence CMPLHSRVPFP (SEQ ID NO: 52);

(b) a polypeptide comprising the sequence QRPVNLTMRRKLRKHNC (SEQ ID NO:53);

(c) a polypeptide comprising the sequenceQRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP (SEQ ID NO: 2); and

(d) an ELABELA polypeptide comprising the sequence of any of SEQ ID NOs:1-36.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibodies or antigen-binding fragments thereoffurther comprise a label.

For the purposes of this document, the term “antibody” refers tocomplete antibodies or antibody fragments capable of binding to aselected target. Unless specified to the contrary, the term includes butis not limited to, polyclonal, monoclonal, natural or engineeredantibodies including chimeric, CDR-grafted and humanised antibodies, andartificially selected antibodies produced using phage display oralternative techniques. The term also includes single chain, Fabfragments and fragments produced by a Fab expression library. Suchfragments include fragments of whole antibodies which retain theirbinding activity for a target substance, Fv, F(ab′) and F(ab′)₂fragments, as well as single chain antibodies (scFv), fusion proteinsand other synthetic proteins which comprise the antigen-binding site ofthe antibody. Small fragments, such as Fv and ScFv, possess advantageousproperties for diagnostic and therapeutic applications on account oftheir small size and consequent superior tissue distribution.

The antibodies and fragments thereof can be humanised antibodies, forexample as described in EP-A-239400. Furthermore, antibodies with fullyhuman variable regions (or their fragments), for example, as describedin U.S. Pat. Nos. 5,545,807 and 6,075,181 can also be used.

The anti-ELABELA antibody can comprise a neutralising antibody.Neutralizing antibodies, i.e., those which inhibit any biologicalactivity of ELABELA, can be used for diagnostics and therapeutics.

The antibodies described here can be altered antibodies comprising aneffector protein such as a label. Labels which allow the imaging of thedistribution of the antibody in vivo or in vitro can be used. Suchlabels can be radioactive labels or radioopaque labels, such as metalparticles, which are readily visualisable within an embryo or a cellmass. Moreover, they can be fluorescent labels or other labels which arevisualisable on tissue samples.

Antibodies can be produced by standard techniques, such as byimmunisation or by using a phage display library. Such an antibody canbe capable of binding specifically to the ELABELA protein or homologue,fragment, etc.

Polyclonal Antibodies

If polyclonal antibodies are desired, a selected mammal (e.g., chicken,mouse, rabbit, goat, horse, etc.) can be immunised with an immunogeniccomposition comprising a ELABELA polypeptide or peptide. Depending onthe host species, various adjuvants can be used to increaseimmunological response. Such adjuvants include, but are not limited to,Freund's, mineral gels such as aluminium hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG(Bacilli Calmette-Guerin) and Corynebacterium parvum are potentiallyuseful human adjuvants which can be employed if purified the substanceamino acid sequence is administered to immunologically compromisedindividuals for the purpose of stimulating systemic defence.

Serum from the immunised animal is collected and treated according toknown procedures. If serum containing polyclonal antibodies to anepitope obtainable from a ELABELA polypeptide contains antibodies toother antigens, the polyclonal antibodies can be purified byimmunoaffinity chromatography. Techniques for producing and processingpolyclonal antisera are known in the art. In order that such antibodiescan be made, we also provide ELABELA amino acid sequences or fragmentsthereof haptenised to another amino acid sequence for use as immunogensin animals or humans.

Monoclonal Antibodies

Monoclonal antibodies directed against epitopes obtainable from aELABELA polypeptide or peptide can also be readily produced by oneskilled in the art. The general methodology for making monoclonalantibodies by hybridomas is well known Immortal antibody-producing celllines can be created by cell fusion, and also by other techniques suchas direct transformation of B lymphocytes with oncogenic DNA, ortransfection with Epstein-Barr virus. Panels of monoclonal antibodiesproduced against orbit epitopes can be screened for various properties;i.e., for isotype and epitope affinity.

Monoclonal antibodies can be prepared using any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueoriginally described by Koehler and Milstein (1975 Nature 256:495-497),the trioma technique, the human B-cell hybridoma technique (Kosbor et al(1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci80:2026-2030) and the EBV-hybridoma technique (Cole et al., MonoclonalAntibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc., 1985).

Recombinant DNA technology can be used to improve the antibodies asdescribed here. Thus, chimeric antibodies can be constructed in order todecrease the immunogenicity thereof in diagnostic or therapeuticapplications. Such techniques comprise splicing of mouse antibody genesto human antibody genes to obtain a molecule with appropriate antigenspecificity and biological activity (Morrison et al (1984) Proc NatlAcad Sci 81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takedaet al (1985) Nature 314:452-454). Moreover, immunogenicity can beminimised by humanising the antibodies by CDR grafting [see EuropeanPatent Application 0 239 400 (Winter)] and, optionally, frameworkmodification [EP 0 239 400].

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,779) can be adapted to produce thesubstance specific single chain antibodies.

Antibodies, both monoclonal and polyclonal, which are directed againstepitopes obtainable from a ELABELA polypeptide or peptide areparticularly useful in diagnosis. Monoclonal antibodies, in particular,can be used to raise anti-idiotype antibodies. Anti-idiotype antibodiesare immunoglobulins which carry an “internal image” of the substanceand/or agent against which protection is desired. Techniques for raisinganti-idiotype antibodies are known in the art. These anti-idiotypeantibodies can also be useful in therapy.

Antibodies can also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G andMilstein C (1991; Nature 349:293-299).

Antibody fragments which contain specific binding sites for thepolypeptide or peptide can also be generated. For example, suchfragments include, but are not limited to, the F(ab′)₂ fragments whichcan be produced by pepsin digestion of the antibody molecule and the Fabfragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragments. Alternatively, Fab expression libraries can beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse W D et al (1989) Science256:1275-128 1).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can also be adapted to produce single chain antibodies toELABELA polypeptides. Also, transgenic mice, or other organismsincluding other mammals, can be used to express humanized antibodies.

The above-described antibodies can be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography.

Recombinant Techniques of Antibody Production

Recombinant DNA technology can be used to produce the antibodiesaccording to established procedure, in bacterial or mammalian cellculture. The selected cell culture system can secrete the antibodyproduct.

Therefore, we disclose a process for the production of an antibodycomprising culturing a host, e.g. E. coli or a mammalian cell, which hasbeen transformed with a hybrid vector comprising an expression cassettecomprising a promoter operably linked to a first DNA sequence encoding asignal peptide linked in the proper reading frame to a second DNAsequence encoding said antibody protein, and isolating said protein.

Multiplication of hybridoma cells or mammalian host cells in vitro iscarried out in suitable culture media, which are the customary standardculture media, for example Dulbecco's Modified Eagle Medium (DMEM) orRPMI 1640 medium, optionally replenished by a mammalian serum, e.g.foetal calf serum, or trace elements and growth sustaining supplements,e.g. feeder cells such as normal mouse peritoneal exudate cells, spleencells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin,low density lipoprotein, oleic acid, or the like. Multiplication of hostcells which are bacterial cells or yeast cells is likewise carried outin suitable culture media known in the art, for example for bacteria inmedium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2×YT, or M9Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, orComplete Minimal Dropout Medium.

In vitro production provides relatively pure antibody preparations andallows scale-up to give large amounts of the desired antibodies.Techniques for bacterial cell, yeast or mammalian cell cultivation areknown in the art and include homogeneous suspension culture, e.g. in anairlift reactor or in a continuous stirrer reactor, or immobilised orentrapped cell culture, e.g. in hollow fibres, microcapsules, on agarosemicrobeads or ceramic cartridges.

Large quantities of the desired antibodies can also be obtained bymultiplying mammalian cells in vivo. For this purpose, hybridoma cellsproducing the desired antibodies are injected into histocompatiblemammals to cause growth of antibody-producing tumours. Optionally, theanimals are primed with a hydrocarbon, especially mineral oils such aspristane (tetramethyl-pentadecane), prior to the injection. After one tothree weeks, the antibodies are isolated from the body fluids of thosemammals. For example, hybridoma cells obtained by fusion of suitablemyeloma cells with antibody-producing spleen cells from Balb/c mice, ortransfected cells derived from hybridoma cell line Sp2/0 that producethe desired antibodies are injected intraperitoneally into Balb/c miceoptionally pre-treated with pristane, and, after one to two weeks,ascitic fluid is taken from the animals.

The foregoing, and other, techniques are discussed in, for example,Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat. No. 4,376,110;Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold SpringHarbor, incorporated herein by reference. Techniques for the preparationof recombinant antibody molecules are described in the above referencesand also in, for example, EP 0623679; EP 0368684 and EP 0436597, whichare incorporated herein by reference.

The cell culture supernatants are screened for the desired antibodies,such as by immunofluorescent staining of PGCs or other pluripotentcells, such as ES or EG cells, by immunoblotting, by an enzymeimmunoassay, e.g. a sandwich assay or a dot-assay, or aradioimmunoassay.

For isolation of the antibodies, the immunoglobulins in the culturesupernatants or in the ascitic fluid can be concentrated, e.g. byprecipitation with ammonium sulphate, dialysis against hygroscopicmaterial such as polyethylene glycol, filtration through selectivemembranes, or the like. If necessary and/or desired, the antibodies arepurified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinitychromatography with the antigen, or fragments thereof, or withProtein-A.

Hybridoma cells secreting the monoclonal antibodies are also provided.Hybridoma cells can be genetically stable, secrete monoclonal antibodiesof the desired specificity and can be activated from deep-frozencultures by thawing and recloning.

Also included is a process for the preparation of a hybridoma cell linesecreting monoclonal antibodies directed to the ELABELA polypeptide,characterised in that a suitable mammal, for example a Balb/c mouse, isimmunised with a one or more ELABELA polypeptides, or antigenicfragments thereof; antibody-producing cells of the immunised mammal arefused with cells of a suitable myeloma cell line, the hybrid cellsobtained in the fusion are cloned, and cell clones secreting the desiredantibodies are selected. For example spleen cells of Balb/c miceimmunised with ELABELA are fused with cells of the myeloma cell line PAIor the myeloma cell line Sp2/0-Ag14, the obtained hybrid cells arescreened for secretion of the desired antibodies, and positive hybridomacells are cloned.

We describe a process for the preparation of a hybridoma cell line,characterised in that Balb/c mice are immunised by injectingsubcutaneously and/or intraperitoneally between 10 and 10⁷ and 10⁸ cellsexpressing ELABELA and a suitable adjuvant several times, e.g. four tosix times, over several months, e.g. between two and four months, andspleen cells from the immunised mice are taken two to four days afterthe last injection and fused with cells of the myeloma cell line PAI inthe presence of a fusion promoter, such as polyethylene glycol. Themyeloma cells can be fused with a three- to twentyfold excess of spleencells from the immunised mice in a solution containing about 30% toabout 50% polyethylene glycol of a molecular weight around 4000. Afterthe fusion the cells are expanded in suitable culture media as describedhereinbefore, supplemented with a selection medium, for example HATmedium, at regular intervals in order to prevent normal myeloma cellsfrom overgrowing the desired hybridoma cells.

Recombinant DNAs comprising an insert coding for a heavy chain variabledomain and/or for a light chain variable domain of antibodies directedto ELABELA as described hereinbefore are also disclosed. By definitionsuch DNAs comprise coding single stranded DNAs, double stranded DNAsconsisting of said coding DNAs and of complementary DNAs thereto, orthese complementary (single stranded) DNAs themselves.

Furthermore, DNA encoding a heavy chain variable domain and/or for alight chain variable domain of antibodies directed to ELABELA can beenzymatically or chemically synthesised DNA having the authentic DNAsequence coding for a heavy chain variable domain and/or for the lightchain variable domain, or a mutant thereof. A mutant of the authenticDNA is a DNA encoding a heavy chain variable domain and/or a light chainvariable domain of the above-mentioned antibodies in which one or moreamino acids are deleted or exchanged with one or more other amino acids.The modification(s) can be outside the CDRs of the heavy chain variabledomain and/or of the light chain variable domain of the antibody. Such amutant DNA is also intended to be a silent mutant wherein one or morenucleotides are replaced by other nucleotides with the new codons codingfor the same amino acid(s). Such a mutant sequence is also a degeneratedsequence. Degenerated sequences are degenerated within the meaning ofthe genetic code in that an unlimited number of nucleotides are replacedby other nucleotides without resulting in a change of the amino acidsequence originally encoded. Such degenerated sequences can be usefuldue to their different restriction sites and/or frequency of particularcodons which are preferred by the specific host, particularly E. coli,to obtain an optimal expression of the heavy chain murine variabledomain and/or a light chain murine variable domain.

The term mutant is intended to include a DNA mutant obtained by in vitromutagenesis of the authentic DNA according to methods known in the art.

For the assembly of complete tetrameric immunoglobulin molecules and theexpression of chimeric antibodies, the recombinant DNA inserts codingfor heavy and light chain variable domains are fused with thecorresponding DNAs coding for heavy and light chain constant domains,then transferred into appropriate host cells, for example afterincorporation into hybrid vectors.

Also disclosed are recombinant DNAs comprising an insert coding for aheavy chain murine variable domain of an antibody directed to ELABELAfused to a human constant domain g, for example γ1, γ2, γ3 or γ4, suchas γ1 or γ4. Likewise recombinant DNAs comprising an insert coding for alight chain murine variable domain of an antibody directed to ELABELAfused to a human constant domain κ or λ, such as κ are also disclosed.

In another embodiment, we disclose recombinant DNAs coding for arecombinant polypeptide wherein the heavy chain variable domain and thelight chain variable domain are linked by way of a spacer group,optionally comprising a signal sequence facilitating the processing ofthe antibody in the host cell and/or a DNA coding for a peptidefacilitating the purification of the antibody and/or a cleavage siteand/or a peptide spacer and/or an effector molecule.

The DNA coding for an effector molecule is intended to be a DNA codingfor the effector molecules useful in diagnostic or therapeuticapplications. Thus, effector molecules which are toxins or enzymes,especially enzymes capable of catalysing the activation of prodrugs, areparticularly indicated. The DNA encoding such an effector molecule hasthe sequence of a naturally occurring enzyme or toxin encoding DNA, or amutant thereof, and can be prepared by methods well known in the art.

Antibodies

The terms “antibody” and “immunoglobulin”, as used in this document, canbe employed interchangeably where the context permits. These terminclude fragments of proteolytically-cleaved or recombinantly-preparedportions of an antibody molecule that are capable of selectivelyreacting with or recognising ELABELA or an epitope thereof, such as anepitope of ELABELA bound by 209.

Non limiting examples of such proteolytic and/or recombinant fragmentsinclude Fab, F (ab′) 2, Fab′, Fv fragments, and single chainantibodies(scFv) containing a VL and VH domain joined by a peptidelinker. These Fvs can be covalently or non-covalently linked to formantibodies having two or more binding sites.

By “ScFv molecules” we mean molecules wherein the VH and VL partnerdomains are linked via a flexible oligopeptide. A general review of thetechniques involved in the synthesis of antibody fragments which retaintheir specific binding sites is to be found in Winter & Milstein(1991)Nature 349, 293-299.

Whole antibodies, and F(ab′) 2 fragments are “bivalent”. By “bivalent”we mean that the said antibodies and F(ab′) fragments have two antigencombining sites. In contrast, Fab, Fv, ScFv and dAb fragments aremonovalent having only one antigen combining site.

The anti-ELABELA antibody can comprise a high affinity antibody with anoff rate from 10⁻²s⁻¹ to 10⁻⁴s⁻¹. The off rate can be about 2×10⁻⁴s⁻¹.

The term “off-rate” as used in this document refers to the dissociationrate (k_(off)) of an antibody such as an anti-ELABELA antibody disclosedhere. It can be measured using BIAevaluation software (Pharmacia). A lowoff rate is desirable as it reflects the affinity of an Fab fragment foran antigen.

The term “affinity” is defined in terms of the dissociation rate oroff-rate (k_(off)) of a an antibody such as an anti-ELABELA antibody.The lower the off-rate the higher the affinity that a an antibody suchas an anti-ELABELA antibody has for an antigen such as ELABELA.

The anti-ELABELA antibody can comprise a peptide per se or form part ofa fusion protein.

The anti-ELABELA antibodies described here include any antibody thatcomprise ELABELA binding activity, such as binding ability tointracellular ELABELA or binding to the same epitope bound by 209 as thecase can be.

The anti-ELABELA antibodies also include the entire or whole antibody,whether mouse, humanised or human, such antibody derivatives andbiologically-active fragments. These can include antibody fragments withELABELA binding activity that have amino acid substitutions or havesugars or other molecules attached to amino acid functional groups, etc.

The anti-ELABELA antibody can comprise isolated antibody or purifiedantibody. It can be obtainable from or produced by any suitable source,whether natural or not, or it can be a synthetic anti-ELABELA antibody,a semi-synthetic anti-ELABELA antibody, a derivatised anti-ELABELAantibody or a recombinant anti-ELABELA antibody.

Where the anti-ELABELA antibody is a non-native anti-ELABELA antibody,it can include at least a portion of which has been prepared byrecombinant DNA techniques or an anti-ELABELA antibody produced bychemical synthesis techniques or combinations thereof.

The term “derivative” as used in this document includes chemicalmodification of an anti-ELABELA antibody. Illustrative of suchmodifications would be replacement of hydrogen by an alkyl, acyl, oramino group, for example. The sequence of the anti-ELABELA antibody canbe the same as that of the naturally occurring form or it can be avariant, homologue, fragment or derivative thereof.

Antibody Variable Regions

The term “variable region”, as used in this document, refers to thevariable regions, or domains, of the light chains (VL) and heavy chains(VH) which contain the determinants for binding recognition specificityand for the overall affinity of the antibody against ELABELA (orvariant, homologue, fragment or derivative), as the case can be.

The variable domains of each pair of light (VL) and heavy chains (VH)are involved in antigen recognition and form the antigen binding site.The domains of the light and heavy chains have the same generalstructure and each domain has four framework (FR) regions, whosesequences are relatively conserved, connected by three complementaritydetermining regions (CDRs). The FR regions maintain the structuralintegrity of the variable domain. The CDRs are the polypeptide segmentswithin the variable domain that mediate binding of the antigen.

The term “constant region”, as used in this document, refers to thedomains of the light (CL) and heavy (CH) chain of the antibody (orvariant, homologue, fragment or derivative) which provide structuralstability and other biological functions such as antibody chainassociation, secretion, transplacental mobility, and complement binding,but which are not involved with binding a ELABELA epitope. The aminoacid sequence and corresponding exon sequences in the genes of theconstant region will be dependent upon the species from which it isderived. However, variations in the amino acid sequence leading toallotypes are relatively limited for particular constant regions withina species. An “allotype” is an antigenic determinant (or epitope) thatdistinguishes allelic genes.

The variable region of each chain is joined to the constant region by alinking polypeptide sequence. The linkage sequence is coded by a “J”sequence in the light chain gene, and a combination of a “D” sequenceand a “J” sequence in the heavy chain gene.

Antibody: Variable Region Sequences

Anti-ELABELA antibodies, according to the methods and compositionsdescribed here, can be generated from these variable region sequences bymethods known in the art. For example, the heavy and light chainsequences can be recombined into a constant sequence for a chosenantibody, through recombinant genetic engineering techniques which areknown to the skilled person.

Constant region sequences are known in the art, and are available from anumber of databases, such as the IMGT/LIGM-DB database (described inGiudicelli et al, 2006, Nucleic Acids Research 34(DatabaseIssue):D781-D784 and LeFranc et al (1995) LIGM-DB/IMGT: An IntegratedDatabase of Ig and TcR, Part of the Immunogenetics Database. Annals ofthe New York Academy of Sciences 764 (1), 47-47doi:10.1111/j.1749-6632.1995.tb55805.x) and the IMGT/GENE-DB database(described in Giudicelli et al, 2005, Nucleic Acids Res. 2005 Jan. 1;33(Database issue):D256-61). IMGT/LIGM-DB and IMGT/GENE-DB are part ofthe ImMunoGeneTics Database located at www.ebi.ac.uk/imgt/.

Methods for combining variable regions with given sequences and constantregions to produce whole antibodies are known in the art and aredescribed for example in Example 16 and in Hanson et al., (2006).Respiratory Research, 7:126. Fragments of whole antibodies such as Fv,F(ab′) and F(ab′)₂ fragments or single chain antibodies (scFv) can beproduced by means known in the art.

Using the disclosed sequences and the methods described in theliterature, for example, the heavy and light chains of the variableregion of antibody 209, having the sequences shown above, can betransgenically fused to a mouse IgG constant region sequence to producea mouse monoclonal anti-ELABELA antibody.

Uses

Anti-ELABELA antibodies can be used in method of detecting a ELABELApolypeptide present in biological samples by a method which comprises:(a) providing an anti-ELABELA antibody; (b) incubating a biologicalsample with said antibody under conditions which allow for the formationof an antibody-antigen complex; and (c) determining whetherantibody-antigen complex comprising said antibody is formed.

Suitable samples include extracts from tissues such as brain, breast,ovary, lung, colon, pancreas, testes, liver, muscle and bone tissues orfrom neoplastic growths derived from such tissues. In particular, asample can comprise a tissue such as a colon, lung, squamous cellincluding lip, larynx, vulva, cervix and penis, pancreatic, brain,oesophageal, stomach, bladder, kidney, skin, ovary, prostate andtesticular tissue from an individual suspected to be suffering from arelevant an ELABELA associated disease.

Antibodies can be bound to a solid support and/or packaged into kits ina suitable container along with suitable reagents, controls,instructions and the like.

Antibody Delivery

The antibodies against the ELABELA protein can be delivered into a cellby means of techniques known in the art, for example by the use ofliposomes, polymers, (e.g., polyethylene glycol (PEG),N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers, polyamidoamine(PAMAM) dendrimers, HEMA, linear polyamidoamine polymers etc) etc. Theimmunoglobulins and/or antibodies can also be delivered into cells asprotein fusions or conjugates with a protein capable of crossing theplasma membrane and/or the nuclear membrane. For example, theimmunoglobulin and/or target can be fused or conjugated to a domain orsequence from such a protein responsible for the translocationalactivity. Translocation domains and sequences can include domains andsequences from the HIV-1-trans-activating protein (Tat), DrosophilaAntennapedia homeodomain protein and the herpes simplex-1 virus VP22protein.

Detection and Diagnostic Methods Detection of Expression of ELABELA

We describe methods of detecting the expression of ELABELA, includingELABELA polypeptides, ELABELA nucleic acids and variants, homologues,derivatives and fragments thereof, etc.

ELABELA expression can be detected as a means to determine the quantityof ELABELA or its activity. ELABELA expression can be detected in or ofa cell, such as a stem cell. Detection of ELABELA expression can also beconducted on a sample comprising a cell tissue, an organ or part or allof an organism.

Expression of ELABELA in an ELABELA association condition can bemodulated, such as up-regulated when compared to normal tissue.Accordingly, we provide for a method of diagnosis of an ELABELAassociated condition, comprising detecting modulation of expression ofELABELA, such as modulation or up-regulation of expression of ELABELA ina cell or tissue of an individual.

Detection of ELABELA expression, activity or amount can be used toprovide a method of determining the state of a cell. Thus, a cell ofinterest can be one with high levels of ELABELA expression, activity oramount compared to a normal cell. Similarly, a cell of interest can beone with low levels ELABELA expression, activity or amount compared to anormal cell.

Detection of ELABELA can also be used to determine whether a cell is acell of interest. Thus, a high level of ELABELA expression, amount oractivity of ELABELA in the cell can be detected. Similarly, a low levelof ELABELA expression, amount or activity can also be detected in acell.

It will be appreciated that if the level of ELABELA varies with theaggressiveness of an ELABELA associated condition, that detection ofELABELA expression, amount or activity can also be used to predict asurvival rate of an individual with an ELABELA associated condition,i.e., high levels of ELABELA indicating a lower survival rate orprobability and low levels of ELABELA indicating a higher survival rateor probability, both as compared to individuals or cognate populationswith normal levels of ELABELA. Detection of expression, amount oractivity of ELABELA can therefore be used as a method of prognosis of anindividual with an ELABELA associated condition.

Detection of ELABELA expression, amount or level can be used todetermine the likelihood of success of a particular therapy in anindividual with an ELABELA associated condition.

The diagnostic methods described in this document can be combined withthe therapeutic methods described. Thus, we provide for a method oftreatment, prophylaxis or alleviation of an ELABELA associated conditionin an individual, the method comprising detecting modulation ofexpression, amount or activity of ELABELA in a cell of the individualand administering an appropriate therapy to the individual based on theaggressiveness of the ELABELA associated condition. The therapy cancomprise an anti-ELABELA agent as described elsewhere.

The presence and quantity of ELABELA polypeptides and nucleic acids canbe detected in a sample as described in further detail elsewhere in thisdocument. Thus, the ELABELA associated diseases can be diagnosed bymethods comprising determining from a sample derived from a subject anabnormally decreased or increased expression, amount or activity, suchas a increased expression, amount or activity, of the ELABELApolypeptide or ELABELA mRNA.

The sample can comprise a cell or tissue sample from an organism orindividual suffering or suspected to be suffering from a diseaseassociated with increased, reduced or otherwise abnormal ELABELAexpression, amount or activity, including spatial or temporal changes inlevel or pattern of expression, amount or activity. The level or patternof expression, amount or activity of ELABELA in an organism sufferingfrom or suspected to be suffering from such a disease can be usefullycompared with the level or pattern of expression, amount or activity ina normal organism as a means of diagnosis of disease.

The sample can comprise a cell or tissue sample from an individualsuffering or suspected to be suffering from an ELABELA associatedcondition, such as a tissue or cell sample of any of those tissues orcells.

In some embodiments, an increased level of expression, amount oractivity of ELABELA is detected in the sample. The level of ELABELA canbe increased to a significant extent when compared to normal cells, orcells from an individual known not to be suffering from an ELABELAassociated condition. Such cells can be obtained from the individualbeing tested, or another individual, such as those matched to the testedindividual by age, weight, lifestyle, etc.

In some embodiments, the level of expression, amount or activity ofELABELA is increased by 10%, 20%, 30% or 40% or more. In someembodiments, the level of expression, amount or activity of ELABELA isincreased by 45% or more, such as 50% or more, as judged by cDNAhybridisation.

The expression, amount or activity of ELABELA can be detected in anumber of ways, as known in the art, and as described in further detailelsewhere in this document. Typically, the amount of ELABELA in a sampleof tissue from an individual is measured, and compared with a samplefrom an unaffected individual. Both ELABELA nucleic acid, as well asELABELA polypeptide levels can be measured.

Detection of the amount, activity or expression of ELABELA can be usedto grade an ELABELA associate condition. For example, a high level ofamount, activity or expression of ELABELA can indicate an aggressiveELABELA associate condition. Similarly, a low level of amount, activityor expression of ELABELA can indicate a non-aggressive ELABELA associatecondition.

Levels of ELABELA gene expression can be determined using a number ofdifferent techniques.

Measuring Expression of ELABELA at the RNA Level

ELABELA gene expression can be detected at the RNA level.

In one embodiment therefore, we disclose a method of detecting thepresence of a nucleic acid comprising a ELABELA nucleic acid in asample, by contacting the sample with at least one nucleic acid probewhich is specific for the ELABELA nucleic acid and monitoring saidsample for the presence of the ELABELA nucleic acid. For example, thenucleic acid probe can specifically bind to the ELABELA nucleic acid, ora portion of it, and binding between the two detected; the presence ofthe complex itself can also be detected.

Thus, in one embodiment, the amount of ELABELA nucleic acid in the formof ELABELA mRNA can be measured in a sample. ELABELA mRNA can be assayedby in situ hybridization, Northern blotting and reversetranscriptase—polymerase chain reaction. Nucleic acid sequences can beidentified by in situ hybridization, Southern blotting, single strandconformational polymorphism, PCR amplification and DNA-chip analysisusing specific primers. (Kawasaki, 1990; Sambrook, 1992; Lichter et al,1990; Orita et al, 1989; Fodor et al., 1993; Pease et al., 1994).

ELABELA RNA can be extracted from cells using RNA extraction techniquesincluding, for example, using acid phenol/guanidine isothiocyanateextraction (RNAzol B; Biogenesis), or RNeasy RNA preparation kits(Qiagen). Typical assay formats utilising ribonucleic acid hybridisationinclude nuclear run-on assays, RT-PCR and RNase protection assays(Melton et al., Nuc. Acids Res. 12:7035. Methods for detection which canbe employed include radioactive labels, enzyme labels, chemiluminescentlabels, fluorescent labels and other suitable labels.

Each of these methods allows quantitative determinations to be made, andare well known in the art. Decreased or increased ELABELA expression,amount or activity can therefore be measured at the RNA level using anyof the methods well known in the art for the quantitation ofpolynucleotides. Any suitable probe from a ELABELA sequence, forexample, any portion of a suitable human ELABELA sequence can be used asa probe. Sequences for designing ELABELA probes can include a sequencehaving SEQ ID NO: 37 to 41, or a portion thereof.

Typically, RT-PCR is used to amplify RNA targets. In this process, thereverse transcriptase enzyme is used to convert RNA to complementary DNA(cDNA) which can then be amplified to facilitate detection.

Many DNA amplification methods are known, most of which rely on anenzymatic chain reaction (such as a polymerase chain reaction, a ligasechain reaction, or a self-sustained sequence replication) or from thereplication of all or part of the vector into which it has been cloned.

Many target and signal amplification methods have been described in theliterature, for example, general reviews of these methods in Landegren,U. et al., Science 242:229-237 (1988) and Lewis, R., Genetic EngineeringNews 10:1, 54-55 (1990).

For example, the polymerase chain reaction can be employed to detectELABELA mRNA.

The “polymerase chain reaction” or “PCR” is a nucleic acid amplificationmethod described inter alia in U.S. Pat. Nos. 4,683,195 and 4,683,202.PCR can be used to amplify any known nucleic acid in a diagnosticcontext (Mok et al., 1994, Gynaecologic Oncology 52:247-252).Self-sustained sequence replication (3 SR) is a variation of TAS, whichinvolves the isothermal amplification of a nucleic acid template viasequential rounds of reverse transcriptase (RT), polymerase and nucleaseactivities that are mediated by an enzyme cocktail and appropriateoligonucleotide primers (Guatelli et al., 1990, Proc. Natl. Acad. Sci.USA 87:1874). Ligation amplification reaction or ligation amplificationsystem uses DNA ligase and four oligonucleotides, two per target strand.This technique is described by Wu, D. Y. and Wallace, R. B., 1989,Genomics 4:560. In the Qβ Replicase technique, RNA replicase for thebacteriophage Qβ, which replicates single-stranded RNA, is used toamplify the target DNA, as described by Lizardi et al., 1988,Bio/Technology 6:1197.

A PCR procedure basically involves: (1) treating extracted DNA to formsingle-stranded complementary strands; (2) adding a pair ofoligonucleotide primers, wherein one primer of the pair is substantiallycomplementary to part of the sequence in the sense strand and the otherprimer of each pair is substantially complementary to a different partof the same sequence in the complementary antisense strand; (3)annealing the paired primers to the complementary sequence; (4)simultaneously extending the annealed primers from a 3′ terminus of eachprimer to synthesize an extension product complementary to the strandsannealed to each primer wherein said extension products after separationfrom the complement serve as templates for the synthesis of an extensionproduct for the other primer of each pair; (5) separating said extensionproducts from said templates to produce single-stranded molecules; and(6) amplifying said single-stranded molecules by repeating at least oncesaid annealing, extending and separating steps.

Reverse transcription-polymerase chain reaction (RT-PCR) can beemployed. Quantitative RT-PCR can also be used. Such PCR techniques arewell known in the art, and can employ any suitable primer from a ELABELAsequence.

Alternative amplification technology can also be exploited. For example,rolling circle amplification (Lizardi et al., 1998, Nat Genet 19:225) isan amplification technology available commercially (RCAT™) which isdriven by DNA polymerase and can replicate circular oligonucleotideprobes with either linear or geometric kinetics under isothermalconditions. A further technique, strand displacement amplification (SDA;Walker et al., 1992, Proc. Natl. Acad. Sci. USA 80:392) begins with aspecifically defined sequence unique to a specific target.

Measuring Expression of ELABELA at the Polypeptide Level

ELABELA expression can be detected at the polypeptide level.

In a further embodiment, therefore, ELABELA expression, amount oractivity can be detected by detecting the presence or amount of ELABELApolypeptide in a sample. This can be achieved by using molecules whichbind to ELABELA polypeptide. Suitable molecules/agents which bind eitherdirectly or indirectly to the ELABELA polypeptide in order to detect itspresence include naturally occurring molecules such as peptides andproteins, for example antibodies, or they can be synthetic molecules.

Thus, we disclose a method of detecting the presence of a ELABELApolypeptide by contacting a cell sample with an antibody capable ofbinding the polypeptide and monitoring said sample for the presence ofthe polypeptide.

For example, the ELABELA polypeptide can be detected using ananti-ELABELA antibody. Such antibodies can be made by means known in theart (as described in further detail elsewhere in this document).

Detection of ELABELA can conveniently be achieved by monitoring thepresence of a complex formed between the antibody and the ELABELApolypeptide, or monitoring the binding between the polypeptide and theantibody. Methods of detecting binding between two entities are known inthe art, and include FRET (fluorescence resonance energy transfer),surface plasmon resonance, etc.

Standard laboratory techniques such as immunoblotting as described abovecan be used to detect altered levels of ELABELA protein, as comparedwith untreated cells in the same cell population.

Gene expression can also be determined by detecting changes inpost-translational processing of ELABELA polypeptides orpost-transcriptional modification of ELABELA nucleic acids. For example,differential phosphorylation of ELABELA polypeptides, the cleavage ofELABELA polypeptides or alternative splicing of ELABELA RNA, and thelike can be measured. Levels of expression of gene products such asELABELA polypeptides, as well as their post-translational modification,can be detected using proprietary protein assays or techniques such as2D polyacrylamide gel electrophoresis.

Assay techniques that can be used to determine levels of ELABELA proteinin a sample derived from a host are well-known to those of skill in theart. Antibodies can be assayed for immunospecific binding by any methodknown in the art.

The immunoassays which can be used include but are not limited tocompetitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA, sandwich immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays and protein A immunoassays. Such assays are routine in theart (see, for example, Ausubel et al., eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which isincorporated by reference herein in its entirety).

Accordingly, in some aspects, provided herein are immunoassay kits formeasuring or detecting ELABELA expression, the immunoassay kitscomprising:

-   -   (a) a coating antigen comprising one or more isolated antibodies        or antigen-binding fragments thereof that specifically binds to        one or more of the following:        -   (i) a polypeptide comprising the sequence CMPLHSRVPFP (SEQ            ID NO: 52);        -   (ii) a polypeptide comprising the sequence QRPVNLTMRRKLRKHNC            (SEQ ID NO: 53);        -   (iii) a polypeptide comprising the sequence            QRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP (SEQ ID NO: 2); or        -   (iv) an ELABELA polypeptide comprising the sequence of any            of SEQ ID NOs: 1-36; and    -   (b) instructions for using said coating antigen.

In some embodiments of these aspects and all such aspects describedherein, the isolated antibodies or antigen-binding fragments thereof arelabelled.

In some embodiments of these aspects and all such aspects describedherein, the immunoassay kit further comprises an enzyme labelledreagent, a secondary antibody that specifically binds to the isolatedantibodies or antigen-binding fragments, a solid substrate, or anycombination thereof.

The specimen can be assayed for polypeptides/proteins byimmunohistochemical and immunocytochemical staining (see generallyStites and Ten, Basic and Clinical Immunology, Appleton and Lange,1994), ELISA, RIA, immunoblots, Western blotting, immunoprecipitation,functional assays and protein truncation test. Other assay methodsinclude radioimmunoassays, competitive-binding assays, Western Blotanalysis and ELISA assays.

ELISA assays are well known to those skilled in the art. Both polyclonaland monoclonal antibodies can be used in the assays. Where appropriateother immunoassays, such as radioimmunoassays (RIA) can be used as areknown to those in the art. Available immunoassays are extensivelydescribed in the patent and scientific literature. See, for example,U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987;3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521 as well asSambrook et al, 1992.

Diagnostic Kits

We also provide diagnostic kits for detecting an ELABELA associatedcondition in an individual, or susceptibility to such an ELABELAassociated condition in an individual.

The diagnostic kit can comprise means for detecting expression, amountor activity of ELABELA in the individual, by any means as described inthis document. The diagnostic kit can therefore comprise any one or moreof the following: a ELABELA polynucleotide or a fragment thereof; acomplementary nucleotide sequence to ELABELA nucleic acid or a fragmentthereof; a ELABELA polypeptide or a fragment thereof, or an antibody toa ELABELA, such as comprising an anti-ELABELA antibody against ELABELA,e.g., an anti-peptide antibody human ELABELA antibody.

The diagnostic kit can comprise instructions for use, or other indicia.The diagnostic kit can further comprise means for treatment orprophylaxis of an ELABELA associated condition, such as any of thecompositions described in this document, or any means known in the artfor treating such an ELABELA associated condition. In particular, thediagnostic kit can comprise an anti-ELABELA agent as described, forexample obtained by screening. The diagnostic kit can comprise atherapeutic drug. The therapeutic drug can also comprise an anti-ELABELAantibody.

Prophylactic and Therapeutic Methods

We disclose methods of treating an abnormal condition, such as anELABELA associated condition, related to insufficient or excessiveamounts of ELABELA expression or activity. Methods of preventing anELABELA associated condition (i.e., prophylaxis) also suitably employthe same or similar approaches.

In general terms, our methods involve manipulation of cells, bymodulating (such as down-regulating) the expression, amount or activityof ELABELA in the cell. A step of detecting modulated ELABELAexpression, amount or activity in a cell can be conducted before orafter the manipulation step. The detection step can detect up-regulatedor down-regulated ELABELA expression, amount or activity. Any of themethods of modulating or down-regulating ELABELA, as described in detailelsewhere in this document, can be used.

The method can comprise exposing the cell to a suitable siRNA, shRNA orchimera RNAi. Examples of siRNA and shRNA are set out in the sequencelisting. For example, any of the shRNA sequences shown as SEQ ID NO: 47to SEQ ID NO: 51 can be employed to down-regulate ELABELA mRNAexpression.

Chimera RNA interference (chimera RNAi) is process by which smallinterfering RNA/DNA chimera triggers the destruction of mRNA for theoriginal gene Chimer RNAi is described in detail in Ui-Tei K et al.,2008, Nucleic Acids Res., April 2008; 36: 2136-2151, Naito al. NucleicAcids Res., July 2005; 33: W589-W591, Ui-Tei K et al., 2004, NucleicAcids Res. 2004 Feb. 9; 32(3):936-48 and Naito et al. Nucleic Acids Res.2004 Jul. 1; 32 (Web Server issue):W124-9.

The method can also comprise exposing the cell to an anti-ELABELAantibody capable of specifically binding to ELABELA. Such an antibodycan comprise any anti-ELABELA antibody, as described elsewhere in thisdocument.

Where ELABELA is associated with aggressiveness of an ELABELA associatedcondition, the level of ELABELA can be detected in a cell of anindividual with an ELABELA associated condition, and the aggressivenessof the ELABELA associated condition assessed. A high level of ELABELAamount, expression or activity compared with a normal cell can indicatean aggressive an ELABELA associated condition, and a stronger or harshertherapy can therefore be required and chosen. Similarly, a lower levelcan indicate a less aggressive therapy.

The approaches described here can be used for therapy of any ELABELArelated disease in general. ELABELA related diseases are described indetail elsewhere in this document.

A ELABELA related disease is defined as being “treated” if a conditionassociated with the disease is significantly inhibited (i.e., by 50% ormore) relative to controls. The inhibition can be by at least 75%relative to controls, such as by 90%, by 95% or 100% relative tocontrols. By the term “treatment” we mean to also include prophylaxis oralleviation of an ELABELA associated condition.

One possible approach for therapy of an ELABELA associated conditions ordisorders is to express anti-sense constructs directed against ELABELApolynucleotides as described here, and administering them to cells orindividuals suffering from an ELABELA associated condition.

Anti-sense constructs can be used to inhibit gene function to preventgrowth or progression in a proliferative cell. Antisense constructs,i.e., nucleic acid, such as RNA, constructs complementary to the sensenucleic acid or mRNA, are described in detail in U.S. Pat. No. 6,100,090(Monia et al.), and Neckers et al., 1992, Crit Rev Oncog 3(1-2):175-231,the teachings of which document are specifically incorporated byreference.

In a particular example, an ELABELA associated condition can be treatedor prevented by reducing the amount, expression or activity of ELABELAin whole or in part, for example by siRNAs capable of binding to anddestroying ELABELA mRNA. We specifically provide for an anti-ELABELAagent which downregulates ELABELA by RNA interference. The anti-ELABELAagent can comprise a Small Interfering RNA (siRNA) or Short Hairpin RNA(shRNA).

RNA interference (RNAi) is a method of post transcriptional genesilencing (PTGS) induced by the direct introduction of double-strandedRNA (dsRNA) and has emerged as a useful tool to knock out expression ofspecific genes in a variety of organisms. RNAi is described by Fire etal., Nature 391:806-811 (1998). Other methods of PTGS are known andinclude, for example, introduction of a transgene or virus. Generally,in PTGS, the transcript of the silenced gene is synthesised but does notaccumulate because it is rapidly degraded. Methods for PTGS, includingRNAi are described, for example, in the Ambion.com world wide web site,in the directory “/hottopics/”, in the “rnai” file.

Suitable methods for RNAi in vitro are described herein. One such methodinvolves the introduction of siRNA (small interfering RNA). Currentmodels indicate that these 21-23 nucleotide dsRNAs can induce PTGS.Methods for designing effective siRNAs are described, for example, inthe Ambion web site described above. RNA precursors such as ShortHairpin RNAs (shRNAs) can also be encoded by all or a part of theELABELA nucleic acid sequence.

Alternatively, double-stranded (ds) RNA is a powerful way of interferingwith gene expression in a range of organisms that has recently beenshown to be successful in mammals (Wianny and Zernicka-Goetz, 2000, NatCell Biol 2:70-75). Double stranded RNA corresponding to the sequence ofa ELABELA polynucleotide can be introduced into or expressed in oocytesand cells of a candidate organism to interfere with ELABELA activity.

Other methods of modulating ELABELA gene expression are known to thoseskilled in the art and include dominant negative approaches. Thus,another approach is to use non-functional variants of ELABELApolypeptide in this document that compete with the endogenous geneproduct resulting in inhibition of function.

One example of a non-functional variant of ELABELA is a mutation toalanine or glycine of an arginine or lysine residue at a position 31 or32—or both—of (or corresponding to) the human ELABELA sequence SEQ IDNO: 20.

ELABELA gene expression can also be modulated by as introducing peptidesor small molecules which inhibit gene expression or functional activity.Thus, compounds identified by the assays described here as binding to ormodulating, such as down-regulating, the amount, activity or expressionof ELABELA polypeptide can be administered to cells to prevent thefunction of ELABELA polypeptide. Such a compound can be administeredalong with a pharmaceutically acceptable carrier in an amount effectiveto down-regulate expression or activity ELABELA, or by activating ordown-regulating a second signal which controls ELABELA expression,activity or amount, and thereby alleviating the abnormal condition.

Suitable antibodies against ELABELA polypeptide as described herein canalso be used as therapeutic agents.

Alternatively, gene therapy can be employed to control the endogenousproduction of ELABELA by the relevant cells such as stem cells in thesubject. For example, a polynucleotide encoding a ELABELA siRNA or aportion of this can be engineered for expression in a replicationdefective retroviral vector, as discussed elsewhere in this document.The retroviral expression construct can then be isolated and introducedinto a packaging cell transduced with a retroviral plasmid vectorcontaining RNA encoding an anti-ELABELA siRNA such that the packagingcell now produces infectious viral particles containing the sequence ofinterest. These producer cells can be administered to a subject forengineering cells in vivo and regulating expression of the ELABELApolypeptide in vivo. For overview of gene therapy, see Chapter 20, GeneTherapy and other Molecular Genetic-based Therapeutic Approaches, (andreferences cited therein) in Human Molecular Genetics, T Strachan and AP Read, BIOS Scientific Publishers Ltd (1996).

In some embodiments, the level of ELABELA is decreased in a stem cell.Furthermore, in such embodiments, treatment can be targeted to, orspecific to, stem cells. The expression of ELABELA can be specificallydecreased only in diseased stem cells, and not substantially in othernon-diseased stem cells. In these methods, expression of ELABELA can benot substantially reduced in other cells, i.e., cells which are not stemcells. Thus, in such embodiments, the level of ELABELA remainssubstantially the same or similar in non-stem cells in the course of orfollowing treatment.

Stem cell specific reduction of ELABELA levels can be achieved bytargeted administration, i.e., applying the treatment only to the stemcells and not other cells. However, in other embodiments,down-regulation of ELABELA expression in stem cells (and notsubstantially in other cell or tissue types) is employed. Such methodscan advantageously make use of stem specific expression vectors, forstem specific expression of for example siRNAs, as known in the art.

Elabela Related Conditions

“ELABELA related condition”, as the term is used in this document, isintended to encompass any cardiac dysfunction, hypertension, or acardiovascular anomaly in blood pressure, cardiac contractility or fluidbalance.

The term “ELABELA related condition” is also intended to encompass anycardiovascular disease such as cardiac hypertrophy, coronary arterydisease (CAD), atherosclerosis, post-infarct treatment, myocardialischemia-reperfusion injury or atrial fibrillation, coronary heartdisease, heart failure, pulmonary arterial hypertension (PAH).

“ELABELA related condition” can also include a condition associated withhigh blood pressure, such as hypertension, angina, congestive heartfailure or erectile dysfunction.

“ELABELA related condition” can also include a condition associated withHIV infection, such as AIDS in an individual.

For example, the methods and compositions described here can be used toprevent, treat or alleviate any of the conditions or diseases set outbelow:

Heart Disease

Heart disease is an umbrella term for a variety for different diseasesaffecting the heart. As of 2007, it is the leading cause of death in theUnited States, England, Canada and Wales, killing one person every 34seconds in the United States alone. Heart disease includes any of thefollowing.

Coronary Heart Disease

Coronary artery disease is a disease of the artery caused by theaccumulation of atheromatous plaques within the walls of the arteriesthat supply the myocardium. Angina pectoris (chest pain) and myocardialinfarction (heart attack) are symptoms of and conditions caused bycoronary heart disease. Over 459,000 Americans die of coronary heartdisease every year. In the United Kingdom, 101,000 deaths annually aredue to coronary heart disease.

Cardiomyopathy

Cardiomyopathy is the deterioration of the function of the myocardium(i.e., the actual heart muscle) for any reason. People withcardiomyopathy are often at risk of arrhythmia and/or sudden cardiacdeath. Extrinsic cardiomyopathies—cardiomyopathies where the primarypathology is outside the myocardium itself comprise the majority ofcardiomyopathies. By far the most common cause of a cardiomyopathy isischemia.

The World Health Organization includes as specific cardiomyopathies:Alcoholic cardiomyopathy, coronary artery disease, congenital heartdisease, nutritional diseases affecting the heart, ischemic (orischaemic) cardiomyopathy, hypertensive cardiomyopathy, valvularcardiomyopathy, inflammatory cardiomyopathy.

Also included are:

Cardiomyopathy secondary to a systemic metabolic disease

Intrinsic cardiomyopathies (weakness in the muscle of the heart that isnot due to an identifiable external cause)

Dilated cardiomyopathy (DCM, the most common form, and one of theleading indications for heart transplantation. In DCM the heart(especially the left ventricle) is enlarged and the pumping function isdiminished)

Hypertrophic cardiomyopathy (HCM or HOCM, a genetic disorder caused byvarious mutations in genes encoding sarcomeric proteins. In HCM theheart muscle is thickened, which can obstruct blood flow and prevent theheart from functioning properly).

Arrhythmogenic right ventricular cardiomyopathy (ARVC, which arises froman electrical disturbance of the heart in which heart muscle is replacedby fibrous scar tissue. The right ventricle is generally most affected)

Restrictive cardiomyopathy (RCM, which is the least commoncardiomyopathy. The walls of the ventricles are stiff, but can not bethickened, and resist the normal filling of the heart with blood).

Noncompaction Cardiomyopathy—the left ventricle wall has failed toproperly grow from birth and such has a spongy appearance when viewedduring an echocardiogram.

Cardiovascular Disease

Cardiovascular disease is any of a number of specific diseases thataffect the heart itself and/or the blood vessel system, especially theveins and arteries leading to and from the heart. Research on diseasedimorphism suggests that women who suffer with cardiovascular diseaseusually suffer from forms that affect the blood vessels while menusually suffer from forms that affect the heart muscle itself. Known orassociated causes of cardiovascular disease include diabetes mellitus,hypertension, hyperhomocysteinemia and hypercholesterolemia.

Types of cardiovascular disease include atherosclerosis

Ischaemic Heart Disease

Ischaemic heart disease is disease of the heart itself, characterized byreduced blood supply to the organs. This occurs when the arteries thatsupply the oxygen and the nutrients gets stopped and the heart will notget enough of the oxygen and the nutrients and will eventually stopbeating.

Heart Failure

Heart failure, also called congestive heart failure (or CHF), andcongestive cardiac failure (CCF), is a condition that can result fromany structural or functional cardiac disorder that impairs the abilityof the heart to fill with or pump a sufficient amount of bloodthroughout the body. Cor pulmonale is a failure of the right side of theheart.

Hypertensive Heart Disease

Hypertensive heart disease is heart disease caused by high bloodpressure, especially localised high blood pressure. Conditions that canbe caused by hypertensive heart disease include: left ventricularhypertrophy, coronary heart disease, (Congestive) heart failure,hypertensive cardiomyopathy, cardiac arrhythmias, inflammatory heartdisease, etc.

Inflammatory heart disease involves inflammation of the heart muscleand/or the tissue surrounding it. Endocarditis comprises inflammation ofthe inner layer of the heart, the endocardium. The most commonstructures involved are the heart valves. Inflammatory cardiomegaly.Myocarditis comprises inflammation of the myocardium, the muscular partof the heart.

Valvular Heart Disease

Valvular heart disease is disease process that affects one or morevalves of the heart. The valves in the right side of the heart are thetricuspid valve and the pulmonic valve. The valves in the left side ofthe heart are the mitral valve and the aortic valve. Included are aorticvalve stenosis, mitral valve prolapse and valvular cardiomyopathy.

[The above text is adapted from Heart disease. (2009, February 3). InWikipedia, The Free Encyclopedia. Retrieved 06:33, Feb. 20, 2009, fromhttp://en.wikipedia.org/w/index.php?fitle=Heart_disease&oldid=268290924]

Pharmaceutical Compositions and Administration

While it is possible for the anti-ELABELA agent, including an ELABELAnucleic acid, polypeptide, fragment, homologue, variant or derivativethereof, modulator, agonist or antagonist, a structurally relatedcompound, or an acidic salt of either to be administered alone, theactive ingredient can be formulated as a pharmaceutical formulation.

We therefore also disclose pharmaceutical compositions comprising ananti-ELABELA agent. Such pharmaceutical compositions are useful fordelivery of the anti-ELABELA agent such as in the form of a compositionas described, to an individual for the treatment or alleviation ofsymptoms as described.

A pharmaceutical composition in the context of the present document is acomposition of matter comprising at least an anti-ELABELA agent as anactive ingredient.

The pharmaceutical formulations comprise an effective amount of theanti-ELABELA agent together with one or more pharmaceutically-acceptablecarriers. An “effective amount” is the amount sufficient to alleviate atleast one symptom of a disease as described.

The effective amount will vary depending upon the particular disease orsyndrome to be treated or alleviated, as well as other factors includingthe age and weight of the patient, how advanced the disease etc stateis, the general health of the patient, the severity of the symptoms, andwhether the anti-ELABELA agent is being administered alone or incombination with other therapies.

Suitable pharmaceutically acceptable carriers are well known in the artand vary with the desired form and mode of administration of thepharmaceutical formulation. For example, they can include diluents orexcipients such as fillers, binders, wetting agents, disintegrators,surface-active agents, lubricants and the like. Typically, the carrieris a solid, a liquid or a vaporizable carrier, or a combination thereof.Each carrier should be “acceptable” in the sense of being compatiblewith the other ingredients in the formulation and not injurious to thepatient. The carrier should be biologically acceptable without elicitingan adverse reaction (e.g. immune response) when administered to thehost.

The active ingredient(s) of a pharmaceutical composition is contemplatedto exhibit therapeutic activity, for example, in the alleviation of anELABELA associated condition. Dosage regimes can be adjusted to providethe optimum therapeutic response. For example, several divided doses canbe administered daily or the dose can be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The active compound can be administered in a convenient manner such asby the oral, intravenous (where water soluble), intramuscular,subcutaneous, intranasal, intradermal or suppository routes orimplanting (e.g. using slow release molecules). Depending on the routeof administration, the active ingredient can be required to be coated ina material to protect said ingredients from the action of enzymes, acidsand other natural conditions which can inactivate said ingredient.

The anti-ELABELA agent can be administered alone, or in combination withother therapeutic agents. Other therapeutic agents suitable for useherein are any compatible drugs that are effective for the intendedpurpose, or drugs that are complementary to the agent formulation. Theformulation utilized in a combination therapy can be administeredsimultaneously, or sequentially with other treatment, such that acombined effect is achieved.

Oral Administration

In some embodiments, the inhibitor of ELABELA activity, expression oramount is provided as an oral composition and administered accordingly.The dosage of the inhibitor of ELABELA activity, expression or amountcan be between about 1 mg/day to about 10 mg/day.

The pharmaceutical composition can be administered in an oralformulation in the form of tablets, capsules or solutions. An effectiveamount of the oral formulation is administered to patients 1 to 3 timesdaily until the symptoms of the disease alleviated.

The effective amount of agent depends on the age, weight and conditionof a patient. In general, the daily oral dose of agent is less than 1200mg, and more than 100 mg. The daily oral dose can be about 300-600 mg.Oral formulations are conveniently presented in a unit dosage form andcan be prepared by any method known in the art of pharmacy. Thecomposition can be formulated together with a suitable pharmaceuticallyacceptable carrier into any desired dosage form. Typical unit dosageforms include tablets, pills, powders, solutions, suspensions,emulsions, granules, capsules, suppositories. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the agent composition with liquid carriers or finely dividedsolid carriers or both, and as necessary, shaping the product. Theactive ingredient can be incorporated into a variety of basic materialsin the form of a liquid, powder, tablets or capsules to give aneffective amount of active ingredient to treat the disease.

The composition can be suitably orally administered, for example, withan inert diluent or with an assimilable edible carrier, or it can beenclosed in hard or soft shell gelatin capsules, or it can be compressedinto tablets, or it can be incorporated directly with the food of thediet. For oral therapeutic administration, the active compound can beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. The amount of active compound in such therapeuticallyuseful compositions in such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like can also contain thefollowing: a binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin can be added or a flavouring agent such aspeppermint, oil of wintergreen, or cherry flavouring. When the dosageunit form is a capsule, it can contain, in addition to materials of theabove type, a liquid carrier.

Various other materials can be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets,pills, or capsules can be coated with shellac, sugar or both. A syrup orelixir can contain the active compound, sucrose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavouring such ascherry or orange flavour. Of course, any material used in preparing anydosage unit form should be pharmaceutically pure and substantiallynon-toxic in the amounts employed. In addition, the active compound canbe incorporated into sustained-release preparations and formulations.

Injectable or Intravenous Administration

In some embodiments, the anti-ELABELA agent is provided as an injectableor intravenenous composition and administered accordingly. The dosage ofthe anti-ELABELA agent inhibitor can be between about 5 mg/kg/2 weeks toabout 10 mg/kg/2 weeks. The anti-ELABELA agent inhibitor can be providedin a dosage of between 10-300 mg/day, such as at least 30 mg/day, lessthan 200 mg/day or between 30 mg/day to 200 mg/day.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene gloycol, and the like), suitablemixtures thereof, and vegetable oils. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of superfactants.

Topical Administration

The pharmaceutical compositions disclosed here include those suitablefor topical and oral administration. Topical formulations can be usedwhere the tissue affected is primarily the skin or epidermis (forexample, psoriasis, eczema and other epidermal diseases).

The topical formulations include those pharmaceutical forms in which thecomposition is applied externally by direct contact with the skinsurface to be treated. A conventional pharmaceutical form for topicalapplication includes a soak, an ointment, a cream, a lotion, a paste, agel, a stick, a spray, an aerosol, a bath oil, a solution and the like.Topical therapy is delivered by various vehicles, the choice of vehiclecan be important and generally is related to whether an acute or chronicdisease is to be treated. As an example, an acute skin proliferationdisease generally is treated with aqueous drying preparations, whereaschronic skin proliferation disease is treated with hydratingpreparations. Soaks are the easiest method of drying acute moisteruptions. Lotions (powder in water suspension) and solutions(medications dissolved in a solvent) are ideal for hairy andintertriginous areas. Ointments or water-in-oil emulsions, are the mosteffective hydrating agents, appropriate for dry scaly eruptions, but aregreasy and depending upon the site of the lesion sometimes undesirable.As appropriate, they can be applied in combination with a bandage,particularly when it is desirable to increase penetration of the agentcomposition into a lesion. Creams or oil-in-water emulsions and gels areabsorbable and are the most cosmetically acceptable to the patient.(Guzzo et al, in Goodman & Gilman's Pharmacological Basis ofTherapeutics, 9th Ed., p. 1593-15950 (1996)). Cream formulationsgenerally include components such as petroleum, lanolin, polyethyleneglycols, mineral oil, glycerin, isopropyl palmitate, glyceryl stearate,cetearyl alcohol, tocopheryl acetate, isopropyl myristate, lanolinalcohol, simethicone, carbomen, methylchlorisothiazolinone,methylisothiazolinone, cyclomethicone and hydroxypropyl methylcellulose,as well as mixtures thereof.

Other formulations for topical application include shampoos, soaps,shake lotions, and the like, particularly those formulated to leave aresidue on the underlying skin, such as the scalp (Arndt et al, inDermatology In General Medicine 2:2838 (1993)).

In general, the concentration of the composition in the topicalformulation is in an amount of about 0.5 to 50% by weight of thecomposition, such as about 1 to 30%, about 2-20%, or about 5-10%. Theconcentration used can be in the upper portion of the range initially,as treatment continues, the concentration can be lowered or theapplication of the formulation can be less frequent. Topicalapplications are often applied twice daily. However, once-dailyapplication of a larger dose or more frequent applications of a smallerdose can be effective. The stratum corneum can act as a reservoir andallow gradual penetration of a drug into the viable skin layers over aprolonged period of time.

In a topical application, a sufficient amount of active ingredient mustpenetrate a patient's skin in order to obtain a desired pharmacologicaleffect. It is generally understood that the absorption of drug into theskin is a function of the nature of the drug, the behaviour of thevehicle, and the skin. Three major variables account for differences inthe rate of absorption or flux of different topical drugs or the samedrug in different vehicles; the concentration of drug in the vehicle,the partition coefficient of drug between the stratum corneum and thevehicle and the diffusion coefficient of drug in the stratum corneum. Tobe effective for treatment, a drug must cross the stratum corneum whichis responsible for the barrier function of the skin. In general, atopical formulation which exerts a high in vitro skin penetration iseffective in vivo. Ostrenga et al (J. Pharm. Sci., 60:1175-1179 (1971)demonstrated that in vivo efficacy of topically applied steroids wasproportional to the steroid penetration rate into dermatomed human skinin vitro.

A skin penetration enhancer which is dermatologically acceptable andcompatible with the agent can be incorporated into the formulation toincrease the penetration of the active compound(s) from the skin surfaceinto epidermal keratinocytes. A skin enhancer which increases theabsorption of the active compound(s) into the skin reduces the amount ofagent needed for an effective treatment and provides for a longerlasting effect of the formulation. Skin penetration enhancers are wellknown in the art. For example, dimethyl sulfoxide (U.S. Pat. No.3,711,602); oleic acid, 1,2-butanediol surfactant (Cooper, J. Pharm.Sci., 73:1153-1156 (1984)); a combination of ethanol and oleic acid oroleyl alcohol (EP 267,617), 2-ethyl-1,3-hexanediol (WO 87/03490); decylmethyl sulphoxide and Azone® (Hadgraft, Eur. J. Drug. Metab.Pharmacokinet, 21:165-173 (1996)); alcohols, sulphoxides, fatty acids,esters, Azone®, pyrrolidones, urea and polyoles (Kalbitz et al,Pharmazie, 51:619-637 (1996));

Terpenes such as 1,8-cineole, menthone, limonene and nerolidol (Yamane,J. Pharmacy & Pharmocology, 47:978-989 (1995)); Azone® and Transcutol(Harrison et al, Pharmaceutical Res. 13:542-546 (1996)); and oleic acid,polyethylene glycol and propylene glycol (Singh et al, Pharmazie,51:741-744 (1996)) are known to improve skin penetration of an activeingredient.

Levels of penetration of an agent or composition can be determined bytechniques known to those of skill in the art. For example,radiolabeling of the active compound, followed by measurement of theamount of radiolabeled compound absorbed by the skin enables one ofskill in the art to determine levels of the composition absorbed usingany of several methods of determining skin penetration of the testcompound. Publications relating to skin penetration studies includeReinfenrath, W G and G S Hawkins. The Weaning Yorkshire Pig as an AnimalModel for Measuring Percutaneous Penetration. In: Swine in BiomedicalResearch (M. E. Tumbleson, Ed.) Plenum, New York, 1986, and Hawkins, G.S. Methodology for the Execution of In Vitro Skin PenetrationDeterminations. In: Methods for Skin Absorption, B W Kemppainen and W GReifenrath, Eds., CRC Press, Boca Raton, 1990, pp. 67-80; and W. G.Reifenrath, Cosmetics & Toiletries, 110:3-9 (1995).

For some applications, a long acting form of agent or composition can beadministered using formulations known in the arts, such as polymers. Theagent can be incorporated into a dermal patch (Junginger, H. E., in ActaPharmaceutica Nordica 4:117 (1992); Thacharodi et al, in Biomaterials16:145-148 (1995); Niedner R., in Hautarzt 39:761-766 (1988)) or abandage according to methods known in the arts, to increase theefficiency of delivery of the drug to the areas to be treated.

Optionally, the topical formulations described here can have additionalexcipients for example; preservatives such as methylparaben, benzylalcohol, sorbic acid or quaternary ammonium compound; stabilizers suchas EDTA, antioxidants such as butylated hydroxytoluene or butylatedhydroxanisole, and buffers such as citrate and phosphate.

Parenteral Administration

The active compound can also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. In someembodiments, the dispersions can be prepared in 30% Capsitol (CyDex,Inc., Lenexa, Kans., USA). Capsitol is a polyanionic β-cyclodextrinderivative with a sodium sulfonate salt separated from the lipophiliccavity by a butyl ether spacer group, or sulfobutylether (SBE). Thecyclodextrin can be SBE7-β-CD.

Adjuvants

The composition can be administered in an adjuvant, co-administered withenzyme inhibitors or in liposomes. Adjuvant is used in its broadestsense and includes any immune stimulating compound such as interferon.Adjuvants contemplated herein include resorcinols, non-ionic surfactantssuch as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.Enzyme inhibitors include pancreatic trypsin. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes.

Prevention of Microorganism Growth

Under ordinary conditions of storage and use, these preparations cancontain a preservative to prevent the growth of microorganisms.

The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thirmerosal, and the like. In manycases, it is possible to include isotonic agents, for example, sugars orsodium chloride. Prolonged absorption of the injectable compositions canbe brought about by the use in the compositions of agents delayingabsorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilisation. Generally, dispersions are prepared byincorporating the sterilised active ingredient into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the methods ofpreparation can include vacuum drying and the freeze-drying techniquewhich yield a powder of the active ingredient plus any additionaldesired ingredient from previously sterile-filtered solution thereof.

Pharmaceutically Acceptable Carrier

As used herein “pharmaceutically acceptable carrier and/or diluent”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, use thereofin the therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

Dosage Unit Forms

It is especially advantageous to formulate pharmaceutical compositionsin dosage unit form for ease of administration and uniformity of dosage.

Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the novel dosage unitforms are dictated by and directly dependent on (a) the uniquecharacteristics of the active material and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such as active material for the treatment of disease inliving subjects having a diseased condition in which bodily health isimpaired.

The principal active ingredients are compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients.

ELABELA Combinations

ELABELA Polypeptides as Disclosed in this Document can be Combined witha Molecule of interest. They can be conjugated to a molecule ofinterest. Alternatively, or in addition, one or more fusion proteins canbe produced comprising an ELABELA polypeptide together with a moleculeof interest such as a polypeptide of interest.

Such combinations are referred to generally in this document as “ELABELAcombinations”.

We disclose an ELABELA combination comprising a first part comprising anELABELA polypeptide and a second part comprising a molecule of interest.The ELABELA polypeptide can comprise any sequence disclosed in thisdocument, for example, a sequence MPLHSRVPFP (SEQ ID NO: 54) orQRPVNLTMRRKLRKHN (SEQ ID NO: 55) or both.

The combination can be such that the first part comprising an ELABELApolypeptide is coupled, fused, mixed, combined, or otherwise joined to asecond part comprising a molecule of interest.

As noted above, the combination can be such that the first part and thesecond part are covalently joined. Such covalent joining can for examplebe achieved by chemical conjugation. Alternatively, or in addition, thecombination can comprise a fusion protein comprising the first part andthe second part, where the molecule of interest comprises a polypeptide.We further disclose an expression construct capable of expressing such afusion protein.

The generation of such combinations of ELABELA polypeptides andmolecules of interest can aid in the tracking, quantitation, extractionor purification of the molecule of interest. In other words, the ELABELApolypeptide can act as a “flag” peptide. Flag peptides are known in theart and include for example glutathione-S-transferase (GST), 6×His (SEQID NO: 97), GAL4 (DNA binding and/or transcriptional activation domains)and β-galactosidase.

The ELABELA combination can be tracked, quantitated, extracted, purifiedetc by means of any agent capable of binding to an ELABELA polypeptide,such as the anti-ELABELA antibodies disclosed elsewhere in thisdocument.

Where ELABELA combinations are produced as fusion proteins, it can alsobe convenient to include a proteolytic cleavage site between the ELABELApolypeptide and the protein sequence of interest so as to allow removalof fusion protein sequences, such as a thrombin cleavage site.

The coupling, etc between the ELABELA polypeptide and the molecule ofinterest in the ELABELA combination can be permanent or transient. Itcan involve covalent or non-covalent interactions (including ionicinteractions, hydrophobic forces, Van der Waals interactions, etc). Theexact mode of coupling is not important. Accordingly, where reference ismade to “comprising”, “conjugation”, “coupling”, etc, these referencesshould be taken to include any form of interaction between the ELABELApolypeptide and the molecule of interest.

ELABELA Fusion Proteins

For example, the ELABELA combination can be a molecule of interest suchas a polypeptide of interest which is provided as a fusion protein withthe ELABELA polypeptide. An expression vector can be constructed bystandard recombinant DNA technology to include a nucleotide sequencecapable of expressing an ELABELA polypeptide together with a nucleotidesequence capable of expressing a polypeptide of interest, such that afusion protein is expressed comprising the ELABELA polypeptide ofinterest fused to the polypeptide of interest. The expression vector canbe transfected or transformed into a suitable host for large scaleproduction of fusion protein, by means known in the art (and asdescribed in detail elsewhere in this document. Purification of thefusion protein can also be carried out by known means.

Alternatively, or in addition, and as discussed above, the ELABELApolypeptide can be physically associated with the molecule of interest,and attached to it by chemical conjugation.

The ELABELA polypeptide of the combination or conjugate or fusionprotein etc can comprise the whole ELABELA molecule, or fragments of it.It can for example comprise the native ELABELA, or any ELABELApolypeptide as disclosed above. The molecule of interest portion cancomprise any molecule of interest, whether proteinaceous or not. Wherethe molecule of interest is proteinaceous in nature (i.e., a polypeptideof interest), it can be conjugated to the ELABELA polypeptide portion bymeans of covalent bonds, for example, amide bonds (for example, as afusion protein).

Furthermore, protein-protein conjugation also provides a convenient andalternative choice for conjugation between the ELABELA polypeptideportion and the molecule of interest portion. Any suitable means ofconjugation, for example, chemical conjugation can be used to couple theELABELA polypeptide and the molecule of interest. Cross-linkers, forexample, heterobifunctional cross linkers are known in the art, and canbe used. Furthermore, other conjugation agents, for example, poly-lacticacid (PLA) and polyethylene glycol (PEG) can also be employed.

Chemical Coupling

As noted above, the ELABELA polypeptide can be coupled to the moleculeof interest by a number of methods. Crosslinkers are divided intohomobifunctional crosslinkers, containing two identical reactive groups,or heterobifunctional crosslinkers, with two different reactive groups.Heterobifunctional crosslinkers allow sequential conjugations,minimizing polymerization.

Any of the homobifunctional or heterobifunctional crosslinkers presentedin the table below can be used to couple the ELABELA polypeptide withthe molecule of interest to produce an ELABELA polypeptide-molecule ofinterest conjugate.

Coupling

The molecule of interest can be attached or coupled to the ELABELApolypeptide by a number of methods. For example, the molecule ofinterest can be coupled to the ELABELA polypeptide by the use ofcyanogen bromide.

Chemical crosslinkers are used to covalently modify proteins forstudying ligand-receptor interactions, conformational changes intertiary structure, or for protein labeling. Crosslinkers are dividedinto homobifunctional crosslinkers, containing two identical reactivegroups, or heterobifunctional crosslinkers, with two different reactivegroups. Heterobifunctional crosslinkers allow sequential conjugations,minimizing polymerization.

Homobifunctional

Modified Reagent code No. Group Solubility Comments Refs BMME 442635-Y—SH DMF, Homobifunctional crosslinker Weston, P. D., et al. Acetoneuseful for formation of 1980. Biochem. conjugates Biophys Acta. 612, viathiol groups. 40. BSOCOES 203851-Y —NH2 Water Base cleavable crosslinkerHoward, A. D., et useful for studying receptors al. 1985. J. Biol. andmapping surface Chem.260, 10833. polypeptide antigens on lymphocytes.DSP 322133-Y —NH2 Water Thiol cleavable crosslinker Lee, W. T., and usedto immobilize proteins Conrad, D. H. 1985. on supports containing aminoJ. Immunol.134, groups. 518. DSS 322131-Y —NH2 Water Non-cleavable,membrane D'Souza, S. E., et impermeable crosslinker al. 1988. J. Biol.widely used for conjugating Chem.263, 3943. radiolabeled ligands to cellsurface receptors and for detecting conformational changes in membraneproteins. EGS 324550-Y —NH2 DMSO Hydroxylamine cleavable Geisler, N., etal. reagent for crosslinking and 1992. Eur. J. reversible Biochem.206,841. immobilization of proteins 14. Moenner, M., et through theirprimary amine al. 1986. Proc. groups. Natl. Acad. Sci. Useful forstudying structure- USA83, 5024. function relationships. EGS, 324551-Y—NH2 Water Water soluble version of EGS Yanagi, T., et al. Water thatreacts rapidly with dilute 1989. Agric. Biol. Soluble proteins atneutral pH. Chem.53, 525. Crosslinked proteins are readily cleaved withhydroxylamine at pH 8.5 for 3-6 hours, 37° C. Glutaral 354400-Y —OHWater Used for crosslinking proteins Harlow, E., and dehyde andpolyhydroxy materials. Lane, D. 1988. Conjugates haptens to carrierAntibodies: A proteins; also used as a tissue Laboratory Manual,fixative. Cold Spring Harbor Publications, N.Y., p. 349. SATA 573100-Y—NH2 DMSO Introduces protected thiols via Duncan, R. J. S., et primaryamines. When treated al. 1983. Anal. with hydroxylamine, yields aBiochem.132, 68. free sulhydryl group that can be conjugated tomaleimide- modified proteins.

Heterobifunctional

Modified Reagent code No. Group Solubility Comments Refs GMBS 442630-Y—NH2, DMSO Heterobifunctional crosslinker Kitagwa, T., et al. —SH usefulfor preparing enzyme- 1983. J. antibody conjugates (for Biochem.94,example beta-gal-IgG) and for 1160.19. Rusin, immobilizing enzymes on K.M., et al. 1992. solid supports. Biosens. Bioelectron.7, 367. MBS442625-Y —NH2, DMSO, Thiol cleavable, Green, N., et al. —SH Waterheterobifunctional reagent 1982. Cell 28, 477. 442626-Y —NH2, especiallyuseful for preparing —SH peptide-carrier conjugates and conjugatingtoxins to antibodies. PMPI 528250-Y —SH2, DMSO, Used in the preparationof Aithal, H. N., et al. —OH DMF alkaline phosphatase 1988. J. Immunol.conjugates of estradiol, Methods112, 63. progesterone, serine-enrichedpeptides, and vitamin B12. SMCC 573114-Y —NH2, DMF, Heterobifunctionalreagent for Annunziato, M.E., —SH AN enzyme labeling of antibodies etal. 1993. 573115-Y —NH2, Acetonitrile and antibody fragments. TheBioconjugate —SH Water cyclohexane bridge provides Chem.4, 212. extrastability to the maleimide group. Ideal reagent for preserving enzymeactivity and antibody specificity after coupling. SPDP 573112-Y —NH2,DMF, Introduces protected thiol Caruelle, D., et al. —SH AN groups toamine groups. 1988. Anal. Acetonitrile Thiolated proteins Biochem.173,328. can be coupled to a second molecule via an iodoacetamide ormaleimide group, or to a second pyridyldisulfide containing molecule.

Each of these reagents can be obtained from a number of manufacturers,for example, from Calbiochem (code No. in column 2), or Piece ChemicalCompany.

The molecule of interest can be activated prior to coupling, to increaseits reactivity. For example, the molecule of interest can be activatedusing chloroacetic acid followed by coupling using EDAC/NHS-OH.Molecules of interest can also be activated using hexane di isocyanateto give primary amino group. Such activated molecule of interest can beused in combination with any hetero bifunctional cross linker. Themolecule of interest in certain embodiments is activated using divinylsulfon. Such activated molecule of interest comprise moieties which canreact with amino or thiol groups, on a peptide, for example.

The molecule of interest can also be activated using tresyl chloride,giving moieties which are capable of reacting with amino or thiolgroups. The molecule of interest can also be activated using cyanogenchloride, giving moieties which can react with amino or thiol groups

Peptide Coupling

The ELABELA polypeptides can be coupled to the molecule of interest bypeptide coupling techniques as described in detail in this section.

Peptides can be obtained by solid phase synthesis methods. The firststage of the technique, first introduced by Merrifield (R. B.Merrifield, Solid Phase Peptide Synthesis. The synthesis of aTetrapeptide., J. Am. Chem. Soc. 85, page 2149-2154, (1963) and R. B.Merrifield, Solid Phase Synthesis, Science 232, page 341-347, (1986))consists of peptide chain assembly with protected amino acid derivativeson a polymeric support. The second stage of the technique is thecleavage of the peptide from the support with the concurrent cleavage ofall side chain protecting groups to give the crude free peptide. Toachieve larger peptides, these processes can be repeated sequentially.

The flexibility of the method allows the synthesis of long, short andbranched peptides, including peptides with natural and un-naturaloccurring amino acids, different linkers and so-called spacers. Thespacers typically being of polyethylenglycol, PEG derivatives orpolyalkanes or homo poly amino acids. The solid phase synthesis methodallows for the preparation of peptides terminated with reactivefunctionalities, for example free thiols, for chemo selective couplingschemes to the molecule of interest material.

A sequence of amino acids can be repeated in the final peptide sequenceto enhance the immunoreactivity with a specific antibody. The repetitiveand reactive sequence can be spaced with irrelevant amino acid sequencesin a linear peptide. Also, by synthesizing branched or dendritic peptideconstructs, like the multiple antigen peptides (MAP), the immunoreactivity can be enhanced.

For a review of the general methodology, including the differentchemical protection schemes and solid and soluble supports, see forexample G. Barany, N. Kneib-Cordonier, D. G. Mullen, Solid-phase peptidesynthesis: A silver anniversary report, Int. J. Peptide Protein Res. 30,page 705-739, (1987), and G. B. Fields, R. L. Noble, Solid phase peptidesynthesis utilizing 9-fluorenylmethoxycarbonyl amino acids, Int. J.Peptide Protein Res. 35, page 161-214 (1990)

Other methods for obtaining peptides include enzymatic fragmentligation, genetic engineering techniques as for example site-directedmutagenesis. Genetic engineering of oligonucleotides, PCR-products, orcloned fragments of DNA material encoding relevant amino acid sequenceusing standard DNA cloning techniques has been a well-establishedmethods of obtaining polypeptides. Alternatively, the peptides can beobtained after isolation from natural sources, such as by proteinpurification and digestion.

Conjugation of the target molecule (for example, peptide) can beachieved by forming covalent bonds or using strong binding pairs, forexample ion binding, biotin-avidin. Examples of other binding entitiesthan streptavidin, avidin and derivatives and biotin and biotinanalogues, are the leucine zipper domain of AP-1 (Jun and fos), hexa-his(SEQ ID NO: 97) (metal chelate moiety), hexa-hat GST (glutathioneS-Transferase) glutathione affinity, trivalent vancomycin, D-Ala-D-Ala,lectines that binds to a diversity of compounds, includingcarbohydrates, lipids and proteins, for example Con A (Canavaliaensiformis), concanavalin A and WGA (Whet germ agglutinin) andtetranectin or Protein A or G. These and other methods are well known toany skilled in the art of conjugation.

Covalent conjugation confers several advantages, including increasedresistance to degradation.

The coupling method useful for conjugation is dependent on the chemicalstructure of the target and the partner involved. Typical chemicalreagents used are so-called zero length cross linkers, homobifunctional,heterobifunctional or polymeric cross linkers.

Zero length cross linkers like 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) or 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimidemetho-p-toluenesulfonate (CHMC) and other carbodiimides can facilitatedirect coupling between for example Glu or Asp to Lysine residues andfor example the N terminus of a peptide.

Homobifunctional cross linkers like glutar(di)aldehydes, imidates,bis-diazotized benzidines, bis(imido esters), bis(succinimidyl esters),diisocyanates, diacid chlorides, divinylsulfone or similar, allows aminoor hydroxyl groups to be bound covalent together through a short linkermolecule. Formaldehyde or glutar(di)aldehyde can also facilitatecross-linking between the ELABELA polypeptide and the molecule ofinterest.

The use of heterobifunctional cross linkers is described in more detailfor cross linking the ELABELA polypeptide to the molecule of interest bya methodology known to any skilled in the art of conjugation.

Heterobifunctional cross linkers have the advantage of providing greatercontrol over the cross-linking than methods which rely on for examplehomobifunctional cross linkers.

The most common schemes for forming a heteroconjugate involve theindirect coupling of an amine group on one bio molecule to a thiol groupon a second bio molecule, usually by a two- or three-step reactionsequence. The high reactivity of thiols and their relative rarity inmany biomolecules make thiol groups ideal targets for controlledchemical cross-linking.

If a thiol group is not present, thiol groups can be introduced byseveral methods. One common method including the use of succinimidyl3-(2-pyridyldithio)propionate (SPDP) followed by reduction of the3-(2-pyridyldithio)propionyl conjugate with DTT or TCEP. Reductionreleases the chromophore 2-pyridinethione, which can be used todetermine the degree of thiolation.

Alternatively, the degree of thiolation can be measured using5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman's reagent) whichstoichiometrically yields the chromophore 5-mercapto-2-nitrobenzoic acidupon reaction with a thiol group.

Heterobifunctional cross linkers typically contain an activated carboxylgroup at one end which can react with amino groups and a maleimido oriodoacetamide group at the opposite end which reacts readily with thesulfhydryl group of cysteine residues.

Two frequently used heterobifunctional crosslinkers areN-gamma-Maleimidobutyryloxysuccinimide ester (GMBS) and Succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carbonate (SMCC).

It should be understood, that the cross linker can contain aphotoactivated reactive moiety. The photo reactive moiety acts as amasked reactive group. By using a photoactive coupling method, it ispossible to bring the target molecule into specific part of for examplethe molecule of interest before using the photo reactive group for thecovalent coupling.

Typically, the peptide is synthesized with a single cysteine residue ateither the N- or C-termini. Alternatively, the internal Cys residues orCys residues on a linker can be used. If the peptide contains no thiolgroup, then one or more can be introduced using one of severalthiolation methods, typically by modifying one of the amino groups.

It should be understood that coupling of target probes, like for examplepeptides, are not limited by the use of thiol selective couplingschemes.

Other useful chemical moieties for both chemo selective or randomconjugation schemes include carboxyl, hydroxyl, aromatic, phenolic oramino groups. Especially amino groups are useful, as they are veryreactive at relevant pH, can form strong chemical bonds and are widelydistributed in biological material.

The possibility to employ conjugation schemes using the amino group inthe N-termini of peptides, including the amino group in the side chainof lysine or polylysine is of special relevance to the compositions andmethods described here.

The cross linker is first reacted with the amino groups on the moleculeof interest, followed by removal of the unreacted cross linker using forexample a decanting or centrifugation. The activated carrier is thenreacted with the Cys-containing peptide. Excess peptide is removed usingfor example a desalting column, dialysis, filtration or centrifugation.The amount of peptide or cross linker attached can be assessed byvarious direct or indirect analytical methods.

The conjugation sequence can be reversed by first attaching theheterobifunctional cross linker to the peptide, before attaching tothiols on the molecule of interest.

During conjugation reaction, the free thiols are often protected againstspontaneous oxidation by the addition of EDTA, EGTA or tributylphosphineor similar or by using a protective atmosphere.

Other methods of covalent cross-linking include the use of homo orheterofunctionel polymeric cross linkers. Examples of reagents includetresyl or vinylsulfone activated dextrans or activated polyacrylic acidpolymers or derivatives. Especially divinyl is preferred for activationof for example hydroxyl groups on the molecule of interest, as theresulting second vinylsulfone is highly reactive towards thiols.

The amount of coupled peptide can be determined by several methods,including incorporating one beta-alanine residue immediately adjacent tothe cysteine residue on the peptide Amino acid analysis can then be usedto determine the amount of beta-alanine present after purification ofthe resulting conjugate.

The cross linkers can offer the possibility to include a tracer ordetectable moiety. This moiety can be used to measure the amount ofcross linker bound to the bio molecule. The tracer can be fluorescent,radioactive, a hapten or any other detectable molecule.

Examples Example 1 Materials and Methods: Accession Codes

The human ELABELA gene and its vertebrate homologs are accessible withthe following Ensembl IDs: Homo sapiens: ENSG00000248329. Mus musculus:ENSMUSG0000007430. Gallus gallus: ENSGALG00000023444. Xenopus laevis: nogene ID exists yet but defined as UniGene X1.40684. Danio rerio:ENSDARG00000094729.

Example 2 Materials and Methods: Cell culture and Assays

The Shef4 cell line was used throughout and exhibits standardmorphological and surface marker characteristics of hESCs and a normal46XY karyotype (Inniss and Moore, 2006). Feeder-free hESCs were culturedin clumps in mTSER1 (Stem Cell Technologies) on Matrigel (BD 354277).

Primary human fibroblasts, SW1353 chondrosarcoma (ATCC) and NTERA2 hECs(a gift from Barbara Knowles and Davor Solter) were grown in Dulbecco'smodified Eagle's medium (DMEM) with 10% FBS (Hyclone), 2 mM GlutaMAX™(Invitrogen) and 1 mM sodium pyruvate.

hESCs were dissociated into single cells using Accutase (Stem CellTechnologies) and plated in the presence of 10 μM Y-27632 (ROCKinhibitor) for 12 hours (Watanabe et al., 2007).

For xCELLigence real time growth assays, 4000 cells were plated per wellof an E-plate (ACEA Biosciences) with media changes every 48 hours.

Recombinant ELA was added at 2.5 uM (or 10 μg/ml). Cell cycle studieswere performed with Click-iT EDU staining kit (Invitrogen) and byperforming a double thymidine block (2.5 mM thymidine; 16 hour block, 8hour release, 16 hour block) followed by DAPI staining for DNA contentat the indicated times following release.

Apoptosis assays were performed by plating control andDoxycycline-treated cells without Y-27632 onto matrigel for 6 hours,followed by harvesting and staining for Annexin V and activated Caspase3.

Embryoid body differentiation was performed by plating 1 million cellsper well of an Aggrewell 400 plate in Aggrewell Medium (Stem CellTechnologies) followed by harvesting and replating into low-adhesionplates (Corning).

After the indicated time periods, embryoid bodies were harvested and RNAextracted using RNeasy kit (Invitrogen). qPCR reactions were carried outusing either Universal FastStart SYBR Green Mastermix (Roche) or usingthe Universal Probe Library system (Roche) in tandem with Taqman FastMastermix (Invitrogen). Primer sequences can be found in Table E1.

TABLE E1 List of qPCR Primers Forward Reverse UPL Transcript (SEQ IDNOs: 98-114) (SEQ ID NOs: 115-131) Probe Human POU5F1 agcaaaacccggaggagtccacatcggcctgtgtatatc 35 ELABELA cacgagtaccctttccctgaggctgggtgtctttccttc 35 NANOG tccagcagatgcaagaactc ttgctattcttcggccagtt87 GATA4 ggaagcccaagaacctgaat gctggagttgctggaagc 69 GATA6aatacttcccccacaacacaa ctctcccgcaccagtcat 90 FOXA2 cgccctactcgtacatctcgagcgtcagcatcttgttgg 9 EOMES gtggggaggtcgaggttc tgttctggaggtccatggtag 6BRA gctgtgacaggtacccaacc catgcaggtgagttgtcagaa 23 NESTINtgcgggctactgaaaagttc tgtaggccctgtttctcctg 76 GAPDH agccacatcgctcagacacgcccaatacgaccaaatcc 60 APLNR atcttgaccctccctggaat atggggagactaggctgtga63 PAX6 aatgggcggagttatgatacc catatcaggttcacttccggg NA SOX17 ABI TaqmanAssay s00751752_s1 NA NKX2.5 ABI Taqman Assay Hs00231763_m1 NA Zebrafishactin gatcttcactccccttgttca ggcagcgatttcctcatc NA aplngctgtgttcagccagtgct ttctgccgcaaaggagtc NA aplnra cgtctgctactgcttcatcggctttttctggtcttccttgc NA aplnrb cctcttgcgctatggacttcgcctgcaatccagtaggtct NA elabela ttcttccacccgctgtatctccggagcatcataaaacctc NA

Example 3 Materials and Methods: shRNA-Mediated ELA Knockdown

To generate stable inducible knockdown of ELA in hESCs, the sequenceGTGATTCTCGTGCCTCAAC (SEQ ID NO: 132) targeting the 3′UTR of ELA wascloned into pSUPERIOR (Oliogoengine) and nucleofected (Lonza) intoShef4_(TetR5) hESCs (Zafarana et al., 2009). Neomycin-resistant cellswere clonally expanded and assayed for doxycycline (20 ng/ml)-inducibleknockdown. Data from one representative clone (Shef4_(TetR5 shELA)) areshown in this paper. Shef4_(TetR5 shβ2M) were derived as previouslydescribed (Zafarana et al., 2009). Unless otherwise stated, ELAknockdown was induced on Day −4 with 20 ng/ml doxycycline to generateshELA cells.

Example 4 Materials and Methods: Antibodies

Polyclonal antibodies were raised against a human N-terminal epitope(nh2-QRPVNLTMRRKLRKHNC) (SEQ ID NO: 53), C-terminal epitope(CMPLHSRVPFP-cooh) (SEQ ID NO: 52) or whole mature ELABELA peptide withan intramolecular cystine bond(nh2-QRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP-cooh) (SEQ ID NO: 2) andpeptide-affinity purified from rabbit and goat sera. For extracellularneutralizing assays, the rabbit α N and α C affinity purified antibodieswere dialysed into PBS overnight at 4° C. and added to the hESCs cultureat 10 μg/mL. For western blotting and immunofluoresence assays, ELAantibodies were used at 1 μg/mL. All other antibodies are commerciallyavailable: TGN-46 (Serotec # AHP500GT); SSEA-3 (Stem Cell Tech#60061AD); TRA-1-60 (BD #560173); SOX17 (R&D # AF1924); POU5F1 (SCBT#sc-5279).

Example 5 Materials and Methods: Embryological Methods

Protocols for fertilization, microinjections, secretion assay andwhole-mount in situ hybridization (WISH) are at our protocol website(http://www.reversade.com-a.googlepages.com/protocols/) and are also setout below.

In Vitro Fertilization of Xenopus Eggs

Nathalie Escande-Beillard, March 2009

This protocol describes the in vitro fertilization of Xenopus eggs, toobtain large amounts of synchronous and dejellyed embryos ready formicroinjections and experimental manipulations.

Day 1—Inducing Ovulation

-   -   1. Proven breeders Xenopus laevis pigmented and albinos are        purchased from Nasco (http://www.enasco.com/xenopus/).    -   2. Choose female's breeders with a big belly and a red cloaca as        these are usually signs of readiness to lay.    -   3. Inject 800 units (800 μl) of Human Chorionic Gonadotropin        (HCG) into the dorsal lymph sac of a female frog. Inject half        volume in each side. Place the needle posteriorly, at the level        of the hindlimb near the lateral line sense organs. Penetrate        the skin with a firm push and then hold the syringe almost        parallel to the back.    -   4. Place the frogs in separate bucket fill with        charcoal-filtered water and cover.    -   5. Frogs kept at room temperature (23° C.) begin laying eggs        about 9-10 hours after induction of ovulation, whereas frogs        kept at 15° C. begin laying eggs approximately 14 hours after        injection.    -   6. After ovulation, frog needs to rest 3 months before being        induced again.

Day 2—In Vitro Fertilization

-   -   7. In the morning, put the female in 1× High Salt Barth's        solution diluted with charcoal filtered water. Keep them in        separate bucket.    -   8. After 1-2 hours start to collect eggs. If the frogs lay well,        it is possible to collect eggs every 1 hour. To collect eggs use        a pipetman outfitted with a 25 ml pipet that has had a little        bit of its tip sawed off with a hacksaw. Always have the        pipetman set to the slow setting while collecting the eggs. As        much as possible try to avoid any perturbations of the eggs at        all points during the process of fertilization. Pipet the eggs        into a 100 mm glass petri dish, trying to limit as much as        possible the amount of buffer, covering about one third the        surface of the plate. Carefully pipet off any extraneous buffer.    -   9. Label glass petri dishes according to which frog the eggs        came from on the side of the bottom dish and the top of the lid.        It is very important to know which frog gives good eggs.    -   10. Sacrifice male and excise testes. Keep testis at 4° C. in        1.5 ml eppendorf tube.    -   11. Draw off as much 1× High Salt Barth as possible using a        Pasteur pipet and tilting the dish. Using Kimwipes, suck up as        much buffer as possible (keeping the dish tilted).    -   12. Add about 1 ml of Steinberg's solution to a 1.5 ml eppendorf        tube. Cut off a small piece of the testis and mince with        scissors in the tube. Add about 5-10 drops of this solution onto        the eggs. Mince fresh testis every time.    -   13. Cover the dish and incubate at room temperature for 5        minutes.    -   14. Carefully add 0.1× Barth. Submerge any floating eggs with        your prewet fingertip. Cover the dish and note the time of        fertilization on the dish lid. Sperm enters eggs as you add 0.1×        Barth. Incubate for 20 minutes at room temperature. Note the        embryos should rotate and the future animal pole (pigmented        side) should face up.    -   15. Decant water, removing excess water with a pasteur pipet.        Add fresh Cysteine Solution, gently pushing submerging any        floating eggs with your finger (put finger into the cysteine        before touching eggs to prevent them from sticking to finger).

Incubate for 8 minutes at room temperature. This step allows thecomplete removal of the jelly surrounding the eggs.

10. Decant and add fresh Cysteine Solution. Incubate 3 minutes.

11. Decant Cysteine Solution and wash at least 6 times with 0.1× Barth.

12. Incubate the eggs in 0.1× Barth. The embryos cultured at roomtemperature should be at 2-cell stage, 1 hour 20 minutes afterfertilization (step #7).

13. Note that embryos are cultured in 0.1× Barth in 50 mm plastic petridishes lined with a cushion of 1% agarose solution.

14. Do not forget to return back female in regular tank at the end ofday 2.

Solutions 10× High Salt Barth's Solution

Fill a 4 L beaker to 3 L with nanopure water and add following:

Final Compound Amount concentration NaCl 256 g 1.095 M KCl 3 g 10 mMNaHCO3 8 g 24 mM MgSO4.7H2O 8 g 8 mM Hepes 95.2 g 100 mM

Add the following dropwise after dissolving each in 25 ml nanopurewater:

Final Compound Amount concentration Ca(NO3)2.4H2O 3.2 g 3.4 mMCaCl2.2H2O 2.4 g 4 mM

pH to 7.7 with HCl. Bring to 4 L with nanopure water. When making a 1×dilution, add 1 ml of 5% Ampicillin per liter.

10× Barth's Solution

Fill 500 ml beaker to 350 ml with nanopure water and add following:

Final Compound Amount concentration NaCl 26 g 889 mM KCl 0.38 g 10 mMNaHCO3 1 g 24 mM Hepes 11.9 g 100 mM

Add the following dropwise after dissolving each in 25 ml nanopurewater:

Final Compound Amount concentration MgSO4.7H2O 1 g 8 mM Ca(NO3)2.4H2O0.4 g 3.3 mM CaCl2.2H2O 0.3 g 4.1 mM

Bring to pH 7.6 with NaOH. Bring to 500 ml with nanopure H2O. Whenmaking the 1× and 0.1× dilutions, add 1 ml of 5% Ampicillin per liter.

10× Steinberg's Solution

Fill 1 liter beaker to 800 ml with nanopure water

Final Compound Amount concentration NaCl 34 g 581 mM KCl 0.5 g 6.7 mMCa(NO3)2.4H2O 0.8 g 3.3. mM MgSO4.7H2O 2 g 8 mM Kanamycin 0.1 g 0.01%Tris Base 6 g 50 mM

pH to 7.35-7.45 with HCl. adjust volume to 1 liter with nanopure H₂O

2% Cysteine Solution

Fill a 1 L beaker to 800 mL with 0.1× Barth's. Add 20 g L-CysteineHydrochloride Monohydrate. pH to 7.8 with NaOH. Bring to 1 liter with0.1× Barth's

5% Ampicillin

Dissolve 5 grams Ampicillin in 100 ml nanopure H2O. Filter sterilize,make 1 ml aliquots in 1.5 ml eppendorf tubes, and store at −20° C.

Preparation of Agarose Petri Dishes

Dissolve Agarose to 1% in water, melt in microwave and add Kanamycin(100 μg/ml). Pour petri dishes with 5 ml of the Agarose solution. Oncecooled down and congealed at room temperature, petri dishes are storedat 4° C.

Chemicals

-   -   i. Human Chorionic Gonadotropin 10 000 units/vial, reconstituted        with 10 ml of water.

Keep at 4° C. Sigma #CG10-IVL.

-   -   ii. Cysteine Hydrochloride Monohydrate. Sigma #C7880-500G.    -   iii. Ampicillin. Sigma #A9518.

Microinjection of Xenopus Embryos

This protocol describes the microinjections of Xenopus embryos for gain-and loss-of-function studies using Morpholinos, DNA, mRNA, Protein orother chemicals.

Day 1—Injections

-   -   i. Embryos for micro-injections are obtained following In Vitro        Fertilization of

Xenopus Eggs described here:http://www.reversade.com-a.googlepages.com/protocols

-   -   ii. Pull needles made of borosilicate glass capillaries using a        micropipette puller. Place pulled needles in safe box as they        are delicate.    -   iii. Link needle to Microinjector with plastic tubing and place        assembled needle onto Micromanipulator.    -   iv. Under a Stereoscopic Microscope calibrate needle by        successively cutting small segments of its fine end with        forceps. 1× Barth solution can be used for calibration purposes.

For cytoplasmic injections calibrate needle so that it delivers 4 nl in1 second with an injection pressure between 20 and 25 PSI. Needles thatare too thin will clog easily and be difficult to fill. Needles that aretoo large will damage embryos upon injection and lead to leakage ofcytoplasm.

4′. For blastocoele injections calibrate needle so that it delivers 50nl in 2 seconds with an injection pressure between 20 and 25 PSI.

-   -   v. Once calibrated, empty and start filling needle with desired        solution.

To fill, carefully place needle into yellow tip containing a few uL ofsolution to inject. Monitor progression of solution inside capillary.Stop before air is drawn.

-   -   vi. Prepare agarose Petri dish filled with 1× Barth. Gently        pipette embryos with eyedropper into 1× Barth Dish. Adjust        magnification, focus, lighting, chair position and rest your        forearms on bunch. You are ready to inject.    -   vii. Bring filled needle close to embryo, hold embryo in        position with forceps, and gently insert needle through chorion        and cytoplasmic membrane. Once inside, inject by pressing foot        pedal linked to Microinjector. In 1 second 4 nL will be        delivered to the embryo. Remove needle by holding embryo.        Microinject each blastomere at the 4-cell stage if all embryo is        targeted.

7′. For blastocoele injections, culture embryo until they reach stage 7to 8. Place in 1× Barth and microinject in blastocoele cavity, situatedjust underneath animal cap. You should see embryo expand as 50 nl aredelivered into blastocoele.

-   -   viii. Once embryos have been injected in 1× Barth, transfer them        into fresh 0.1× Barth dish. Label lid of dish. Do not forget to        keep uninjected embryos. Control embryos should be from the same        fertilization batch and mother frog.    -   ix. Culture embryos in 0.1× Barth at 13° C. (not below) to slow        down development or up to 22° C. for faster development.

Day 2—Look after Your Embryos

-   -   2. Everyday remove dead embryos. De-chorionate using fine        forceps and place embryos in fresh dish filled with 0.1× Barth        using an eyedropper.    -   3. By stage 20, it is preferable to culture embryos in 1×        Steinberg.    -   4. To fix embryos, drop embryos with eyedropper in 2 ml of MEMFA        in glass vial. Fix for 2 hours at room temperature or overnight        at 4° C. with gentle rocking.    -   5. Dehydrate through a methanol series (25%, 50%, 75%, 2×100% in        PBS 1×). Embryos can be stored in 100% methanol at −20° C.    -   6. For Xenopus Whole Mount In Situ Hybridization follow protocol        described here:        http://www.reversade.com-a.googlepages.com/protocols

Solutions 10× Barth's Solution

Fill 500 ml beaker to 350 ml with nanopure water and add following:

Final Compound Amount concentration NaCl 26 g 889 mM KCl 0.38 g 10 mMNaHCO3 1 g 24 mM Hepes 11.9 g 100 mM

Add the following dropwise after dissolving each in 25 ml nanopurewater:

Final Compound Amount concentration MgSO4.7H2O 1 g 8 mM Ca(NO3)2.4H2O0.4 g 3.3 mM CaCl2.2H2O 0.3 g 4.1 mM

Bring to pH 7.6 with NaOH. Bring to 500 ml with nanopure H2O. Whenmaking the 1× and 0.1× dilutions, add 1 ml of 5% Ampicillin per liter.

10× Steinberg's Solution

Fill 1 liter beaker to 800 ml with nanopure water

Final Compound Amount concentration NaCl 34 g 581 mM KCl 0.5 g 6.7 mMCa(NO3)2.4H2O 0.8 g 3.3. mM MgSO4.7H2O 2 g 8 mM Kanamycin 0.1 g 0.01%Tris Base 6 g 50 mM

pH to 7.35-7.45 with HCl. Adjust volume to 1 liter with nanopure H₂O

10×MEM

To make 1 L

209.26 g MOPS (pH 7.4) 100 mM 9.36 g EGTA 2 mM 1.2 g MgSO4 1 mM

Adjust pH to 7.4 with 10 M NaOH, filter and store at 4° C.

MEMFA 1×

Prepare a small volume of 1× each time. For 100 ml:10 ml of 10×MEM, 10ml of Formaldehyde (37%), 80 ml of Water.

Agarose Petri Dishes

Dissolve Agarose to 1% in water, melt in microwave and add Kanamycin(100 μg/ml). Pour Petri dishes with 5 ml of the Agarose solution. Oncecongealed at room temperature, Petri dishes are stored at 4° C.

Instruments

Needle puller: SUTTER INSTRUMENT CO. Flaming/brown Micropipette pullerMODEL P-97

Needles: World Precision Instruments, Inc. Borosilicate GlassCapillaries Item number: TW 100-6

Microinjectors: Harvard APPARATUS PLI-100 OR NARISHIGE IM 300Microinjector

Micromanipulator: Singer Instruments Mk1 Micromanipulator

Stereomicroscope: Leica MZ12.5 or MZ9.5

Light Source: Leica CLS 150×

Forceps: Rustless DUMOXEL #3 or #5 FST by DUMONT Switzerland

Eyedropper: Fisher Scientific Pipet Straight Med Drop FIS#13-700

Glass Vial: Fisher Scientific Vial ST W/Closure 1DR FIS#03-399-25B

Xenopus Whole Mount In Situ Hybridization

ISH is a method used to determine the spatio-temporal expression patternof a gene.

Synthesis of Antisense RNA Probe

-   -   i. Linearize DNA plasmid by digesting with a suitable enzyme.        Check for complete digestion by running 1 μl of DNA on agarose        gel. **All conditions should be RNase free: use gloves and        filtered tips.    -   ii. Phenol:Chloroform extract and ethanol precipitate DNA or use        a Kit for cleaning DNA.    -   iii. Resuspend DNA in a suitable volume of RNase free water to        get approximately 0.5 or 1 μg/μl. Measure DNA concentration        (nanodrop).    -   iv. Set up transcription reaction: 1000-2000 ng DNA, 4 μl 10×        Transcription Buffer, 4 μl Labelling Mix (Digoxigenin), 1 μl RNA        guard (40 U/μl), 4 μl RNA polymerase (20 U/μl), RNAse free Water        to 40 μl    -   v. Incubate for 4-5 hours at 37° C.    -   vi. Treat with DNase for 20 nm at 37° C. (1 unit of enzyme/μg        DNA).    -   vii. Remove unincorporated, free nucleotides with Quick Spin        Columns:        -   1. First remove top cap from column and then bottom cap to            avoid air bubbles.        -   2. Spin for 5 minutes at 4° C., 1800 rpm in a swing bucket            centrifuge.        -   3. Remove eluate and spin for an additional 5 minutes.        -   4. Put columns in new (labeled) tubes, add transcription            reaction making sure to add it directly on the center of the            column while not disrupting resin with the pipette tip.        -   5. Spin for 15 minutes at 4° C., 1800 rpm.    -   viii. Quantify RNA yield with nanodrop to determine how much to        use for the ISH.    -   ix. Run a gel with 1 μl to verify expected size and quality of        probes

General Notes

All recipes and most chemicals used are listed with their vendor andcatalog number at the end of this protocol. Very important to work inRNAse free conditions. Use gloves and filtered tips from the fixationstep to the end of hybridization.

Day 1

All steps in Day 1 are done ON ICE except for Proteinase K treatment(step #3). Use at least 2 ml of buffer for each wash, unless indicated.

-   -   2. Rehydrate the embryos through a methanol series in PBSw (75%,        50%, 25%). Each rehydration step is incubated for 5 minutes.    -   3. Wash 3× with PBSw, 5 minutes each.    -   4. Treat embryos for 8 minutes with 10 μg/ml Proteinase K in        PBSw at room temperature (1 ml per tube). Staining for highly        expressed genes requires less digestion, but longer digestion        can help for genes with lower expression. Do not exceed 8        minutes!    -   5. Stop digestion by washing with 2 mg/ml glycine in PBSw.    -   6. Do a fast wash with PBSw, and then wash 2× with PBSw, 5        minutes each.    -   7. Refix embryos in 5 ml of 4% paraformaldehyde/0.2%        glutaraldehyde in PBSw for 15 minutes. Make the buffer fresh        each time.    -   8. Do a fast wash with PBSw, and then wash 3× with PBSw, 5        minutes each.    -   9. Wash in 1 ml of 50% PBSw/50% Hybridization Solution for 3        minutes.    -   10. Wash in 1 ml of Hybridization Solution (100%) for 3 minutes.        Embryos can also be stored at this step in Hybridization        Solution at −20° C.    -   11. Replace 1 ml Hybridization Solution with 400 μl        Hybridization Solution and prehybridize for 3 hours at 65° C.    -   12. Denature appropriate amount of probe in 100 μl Hybridization        Solution at 95° C. for 5 minutes. Put on ice, vortex and add        this mix to the embryos and hybridize overnight at 70° C.

Day 2

-   -   13. Remove probe/hybridization mix and replace with 800 μl        Hybridization Solution. Wash for 5 minutes at 70° C.    -   14. Add 400 μl 2×SSC (pH4.5) to each vial. Incubate 5 minutes at        70° C.    -   15. Repeat the previous step 2 more times (final volume=800        μl+400 μl+400 μl+400 μl=2000 μl). Incubate at 70° C. for 5        minutes each time.    -   16. Remove the mix and wash 2× with 2×SSC (pH7)/0.1% CHAPS at        70° C. for 30 minutes.    -   17. Wash 2× in MAB at room temperature for 10 minutes each.    -   18. Wash 2× in MAB at 70° C. for 30 minutes each.    -   19. Wash 2× in PBS at room temperature for 10 minutes each.    -   20. Wash in PBSw at room temperature for 5 minutes.    -   21. Incubate the embryos in 1 ml Antibody Buffer (without        antibody) at 4° C., rocking, for a minimum of 2 hours.    -   22. At this time also pre-block the antibody in Antibody Buffer        at 4° C., rocking for 2 hours: Anti-Dig—Alkaline Phosphatase        dilute 1:5000 from a stock of 150 units/200 ul.        Anti-Dig—Peroxidase dilute 1:200 from a stock of 150 units/ml.    -   23. Replace Antibody Buffer with 1.5 ml of pre-blocked antibody.        Incubate with rocking at 4° C. overnight. Check embryos to make        sure all are immersed in solution (not stuck in lid of glass).

Day 3

-   -   24. Fast wash embryos with 0.1% BSA in PBSw.    -   25. Wash 5× in 0.1% BSA in PBSw, with rocking, 1 hour each wash        at room temperature.    -   26. Wash 2× in PBSw for 30 minutes each, at room temperature.    -   27. Wash 2× in AP1 Buffer for BM Purple staining for 10 minutes        each.    -   28. Replace AP1 Buffer with 1 ml BM Purple, cover with Aluminium        foil and incubate with rocking until desired staining is        reached. Check embryos to make sure all are immersed in solution        (not stuck in lid of glass). Staining time will vary depending        on the level of expression and probe quality. It is recommended        to let the reaction take place at 4° C., overnight. At room        temperature embryos will tend to get more background.

Day 4

-   -   29. Stop staining reaction by washing 2 times in Stop Solution        for 15 minutes each. Rinse caps as well.    -   30. Dehydrate through a methanol series (25%, 50%, 75%, 2×100%).        Embryos can be stored in methanol at −20° C.    -   31. Optional: to remove pigmentation or excess background and to        enhance contrast, embryos can be bleached with 3 ml of fresh        bleaching solution. To speed up the process, place tubes on        aluminium foil and under intense neon light several hours to        overnight.    -   32. Put back embryos in methanol 100%.

Solutions

When diluting 10× stock solution use DEPC treated water and filter. Allother buffers are made using DEPC treated water and then filter.

DEPC Treated Buffers or Water

Add 0.1% DEPC. Incubate with agitation until it is completely dissolvedand autoclave.

10×PBS

80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4, 800 ml distilled Water(DDW). Dissolve, pH to 7.4, add DDW to 1 L, DEPC treat and autoclave.

PBSw

PBS with 0.1% Tween-20. DEPC treat and autoclave.

20×SSC

175.3 g NaCl, 88.2 g Sodium Citrate, 800 ml DDW. Dissolve, pH to 7.0,add DDW to 1 L, DEPC treat and autoclave.

4% Paraformaldehyde

Dissolve paraformaldehyde in fresh PBS (4 g for 100 ml). Heat at 60° C.and mix until completely dissolved. Cool on ice, filter, aliquot andstore at −20° C.

Hybridization Solution

Make 1 L, filter, aliquot and store at −20° C. 10 g Boehringer Block,500 ml Formamide, 250 ml 20×SSC. Heat at 65° C. for 1 hour. 120 ml DEPCtreated water, 100 ml Torula RNA (10 mg/ml in water; filtered), 2 mlHeparin (50 mg/ml in 1×SSC), 5 ml 20% Tween-20, 10 ml 10% CHAPS, 10 ml0.5M EDTA

MAB

100 mM Maleic Acid, 150 mM NaCl pH 7.5

Boehringer Blocking Solution (10%)

Dissolve Boehringer blocking reagent in maleic acid buffer (MAB) (10 gfor 100 ml). Heat and vortex frequently to dissolve completely. Store at−20° C. as a stock solution.

Antibody Buffer

10% heat inactivated goat serum, 10% Boehringer blocking stock solution.80% PBSw. Heat at 70° C. for 10 minutes, vortex, cool on ice and filter.Aliquot and store at −20° C.

AP1 Buffer

0.1M NaCl, 0.1M Tris pH 9.5, 50 mM MgCl2

Stop Solution

100 mM Tris pH 7.4, 1 mM EDTA

Bleaching Solution

⅔ Methanol 100%, ⅓ Hydroxyde Peroxyde 31.5% (final 10.5%), Make fresheach time.

Chemicals for Making the Probe

Dig RNA Labelling Mix (10×)—Roche #1277073, Flu RNA Labelling Mix(10×)—Roche #1685619, Transcription Buffer (10×)—Roche #1465384, RNAGuard—Roche #3335399, T3 RNA Polymerase—Roche #1031163, T7 RNAPolymerase—Roche #881767, SP6 RNA Polymerase—Roche #810274, Quick SpinColumns—Roche #1274015, QIAquick Gel Extraction Kit—Qiagen #28706

Chemicals for ISH

Boehringer Block—Roche #1096176, Proteinase K—Gibco #25530-049, AntiDig-AP—Roche #1093274, Anti Flu-AP—Roche #1426338, Anti Dig-POD—Roche#1207733, Anti Flu-POD—Roche #1426346, BM Purple—Roche #1442074,Hydrogen Peroxide 31.5%—Calbiochem#386790.

ZFNs were purchased from Sigma-Aldrich. 250 pg of mRNAs encoding the ZFNpair were injected into 1-cell stage embryos. ZFN binding sites withinexon 1 are as follows: 5′_TCCACCCGCTGTATCT_(—)3′ (SEQ ID NO: 133) and5′_GCTGCTGCTGACAGT_(—)3′ (SEQ ID NO: 134). Sequencing primers flankingthe mutation sites are: forward 5′_AACACTTGCTGAGAGCGACAG_(—)3′ (SEQ IDNO: 135), reverse 5′_AGATGTGGTGGTGTTGAGTAGC_(—)3′ (SEQ ID NO: 136). Forela overexpression, 200 pg of SP6-transcribed zebrafish ela capped mRNA(Applied Biosystems) was injected into 1-cell stage zebrafish embryos.Translation blocking MOs used for aplnra and aplnrb have been describedpreviously (Scott et al., 2007; Zeng et al., 2007) and were purchasedfrom Gene tools (Oregon, USA). Embryos were injected at 1-cell stagewith a combination of 1 ng of aplnra MO and 0.5 ng of aplnrb MO (Zeng etal., 2007). Information on probes used for q-PCR and WISH can be foundin Tables E1 and E2. Zebrafish embryos at 100% epiboly weredechorionated and processed for western blotting as previously described(Link et al., 2006). For the Xenopus secretion assay, 16 ng ofSP6-transcribed human ELA capped mRNA (Applied Biosystems) was injectedinto each cell of 4-cell stage embryos. At stage 8, embryos weredechorionated and dissociated in CMFM medium with 5 mM EDTA. Cellpellets of 10 embryos were transferred into 1.5 mL tubes and allowed tosecrete for 24 hours at room temperature in 40 ml of fresh CMFM mediumwith or without BFA. The conditioned supernatant was carefully depletedof all cells before SDS-PAGE electrophoresis on 16.5% Tris-Tricineprecast gels (BioRad).

ISH Probes

TABLE E2 In situ Hybridisation Probes. Forward Reverse (SEQ ID NOS137-140) (SEQ ID NOS 141-144) aplnra atggagccaacgccggaattcacactttggtggccagc aplnrb atgaatgccatggacaacat tcacaccttcgtagccagccmlc1 acacacatcccagccttttc ccaatttcattcggcaatct ela ccatccctcagaggacagagcatgtttggcagcagtagga References bra (Schulte-Merker et al., 1994) gata5(Reiter et al., 1999) scl (Schoenebeck and Yelon, 2007) sox17 (Alexanderand Stainier, 1999)

Example 6 Materials and Methods: ELISA

For sandwich ELISA assays, goat α C antibody (4 μg/ml) was used as thecapture antibody and blocked with 3% BSA in PBS-Tween (0.05% v/v). hESCswere cultured for 24 hours in mTSER1 and supernatants harvested andincubated with the capture antibody for 1 hour at room temperature.After 5 washes with PBST, rabbit α C (0.8 μg/mL) followed by anti-rabbitHRP (Jackson Immunolabs, 0.12 μg/ml) in 3% BSA/PBST were used fordetection with 3,3′,5,5′-Tetramethylbenzidine chromogenic substrate.Recombinant ELA peptide of known concentration (Pierce BCA Protein Assaykit) was used to generate a standard curve.

Example 7 Materials and Methods: AP-ELA Binding Assay

An AP-ELA fusion construct was generated by cloning the matureC-terminal 32-mer of ELA in frame with the 3′ end of the alkalinephosphatase ORF containing an N-terminal signal sequence from XenopusChordin (Reversade and De Robertis, 2005). This construct wastransfected into 293T cells with Fugene HD (Promega) and allowed tosecrete for 48 hours into serum-free media (Pro293a CDM, Lonza). Theresulting supernatant was incubated with test cells for 3 hours, washed3 times with PBS, lysed, and heated at 65° C. to inactivate endogenousalkaline phosphatases. Lysates were then incubated with BM Purple(Roche) at 37° C. for chromogenic development.

Example 8 Materials and Methods: Statistical Analysis

To analyze the statistical significance of differences between groupmeans, two-tailed unpaired Student's T-test was used throughout thestudy. Significance levels are denoted by * (p<0.05); ** (p<0.01), ***(p<0.001).

Example 9 Materials and Methods: DNA Constructs and Site-DirectedMutagenesis

Zebrafish aplnra, aplnrb and elabela ORFs were cloned from 80%-epibolycDNA into vector pCS2+ between restriction sites BamHI and XbaI. HumanAPLNR and GPR15 ORFs were cloned from human genomic DNA into pCS2+between restriction sites BamHI and XbaI. Primers used can be found onTable E3. aplnrb^(grinch) (aplnb^(w90L)) was generated using QuikChangeSite-directed Mutagenesis kit (Stratagene), with the following primerpair: 5′_CTTTGTGGTGACCCTGCCCCTGTTGGCCGTCTACACTGCTCTG_(—)3′ (SEQ ID NO:145) and 5′_CAGAGCAGTGTAGACGGCCAACAGGGGCAGGGTCACCACAAAG_(—)3′ (SEQ IDNO: 146).

Cloning Primers

TABLE E3 List of cloning primers Forward (SEQ ID NOS 147- Reverse (SEQID NOS 150-151, 148, 137-138 and 149) 142 and 152) Human APLNRatggaggaaggtggtgattttga ggagacccttgtggttgactag GPR15atggacccagaagaaacttcag ttagagtgacacagacctcttcc Zebrafish aplnraatggagccaacgccggaat tcacactttggtggccagc aplnrb atgaatgccatggacaacattcacaccttcgtagccagc ela atgagattcttccacccgc tcaagggaaaggtactctggag

Example 10 Materials and Methods: siRNA-Mediated ELA Knockdown

Anti-ELA siRNAs were produced by RNase III (New England Biolabs)cleavage of double stranded full-length ELA mRNA produced by T7 in vitrotranscription (Applied Biosystems). siRNAs were transfected into hESCswith Lipofectamine RNAiMax (Invitrogen) for 48-72 hours prior to assay.

Example 11 Materials and Methods: POU5F1 Knockdown

POU5F1 is also known as OCT-4. Inducible knockdown of POU5F1 inShef4_(TetR5) was performed as previously reported by Zarafana andcolleagues (Zafarana et al., 2009). Briefly, Shef4_(TetR5 shPOU5F1)hESCs were cultured in the presence (shPOU5F1) or absence (Control) of 5ng/ml doxycycline for the indicated durations and harvested for qPCRanalysis.

Example 12 Materials and Methods: Luciferase Reporter Assay

The ELA promoter situated between coordinateschr4:165,796,806-165,798,359 (hg19) was amplified from human genomic DNAand cloned upstream of a firefly luciferase reporter gene in pGL3basic(Promega) to generate pGL3-ELA_(promoter). To generatepGL3-ELA_(promoter+POU5F1enhancer), the POU5F1 enhancer region situatedbetween coordinates chr4:165,787,570-165,788,797 (hg19) containingseveral POU5F1 putative binding sites as predicted by TRANSFAC (Matys etal., 2006) was cloned from human genomic DNA and inserted 5′ of the ELApromoter in pGL3-ELA_(promoter). These constructs were nucleofected intohESCs (Human Stem Cell Nucleofector® kit, Lonza) together with pRL-CMV(Promega) for 24 hours. Normalized firefly activity was measured usingDual-Glo® Luciferase Assay System (Promega).

Example 13 Materials and Methods: Teratoma Analysis

Shef4_(TetR5-shELA) cells were cultured in the presence (shELA) orabsence (Control) of 20 ng/ml doxycycline for 3 passages (4days/passage). Cells were harvested with Accutase (StemcellTechnologies) and resuspended in 1 part Matrigel to 2 parts mTSER1. 10million cells in 200 μl were injected subcutaneously into the right hindpaunches of SCID mice. shELA-injected mice were fed doxycycline (20μg/ml) for 3 weeks following injection. Mice were sacrificed 60 dayspost-injection for tumor analysis.

Example 14 Materials and Methods: Directed Endoderm Differentiation

Shef4_(TetR5-shELA) and Shef4_(TetR5-shβ2M) cells were cultured in thepresence (shELA and shβ2M respectively) or absence (Control) of 20 ng/mldoxycycline for 1 passage (4 days) and dissociated with Accutase. 0.6million cells were plated into each well of a matrigel-coated 6 welldish in the presence of 10 uM Y-27632 in mTSER1. 24 hours later, themedia was changed to Endo differentiation media 1 [RPMI 1640 (Gibco), 1×B-27 supplement (Invitrogen), 50 ng/ml of Activin A (R&D Systems), 25ng/ml BMP4 (R&D Systems) and 50 ng/ml bFGF (Invitrogen)] for 3 days. Onday 3, the media was changed to Endo differentiation media 2 (RPMI 1640,B27, 50 ng/ml Activin) for 2 more days of culture. Rescue experimentswere performed by adding 2.5 uM of ELA peptide to Shef4_(TeTR5-shELA)concurrently with doxycycline up to day 3, when the ELA peptide is nolonger necessary as judged by downregulation of endogenous ELA.

On day 5, cells were fixed with 4% paraformaldehyde and stained with αSOX17 (AF1924, R&D Systems) and DAPI. For each well, three differentrandomly selected fields were imaged and the number of nuclei stainingpositive of SOX17 and DAPI were counted using a custom macro in ImageJ.A minimum of two wells per condition was used for each experiment, and aminimum of two independent experiments were performed to generate theresults.

Example 15 Materials and Methods: shRNA-Mediated Knockdown of APLNR

Lentiviral constructs encoding shRNA hairpins were prepared aspreviously described (Tiscornia et al., 2006a). Briefly, L-CMV-GFP-NheIvector was modified by replacing the CMV-GFP with PGK-GFP-P2A PUROcassette for stable expression in hESCs. Validated shRNA sequences(shAPLNR-1: 5′ ACACGTACCGGGACTATGA 3′ (SEQ ID NO: 153), shAPLNR-2: 5′CCATCATGCTGACCTGTTA 3′ (SEQ ID NO: 154) and Control: 5′GACCTCTGCGCCTAATTAT 3′ (SEQ ID NO: 155)) were cloned into NheI sitealong with the along with H1 promoter. Lentiviral particles wereprepared as described (Tiscornia et al., 2006b). HES3 human embryonicstem cells were transduced with 2TU of lentiviruses and selected with 2ug/ml puromycin for 2 weeks on feeder free culture system.

Example 16 Materials and Methods: Mesoendoderm Differentiation of hESC

To obtain hESC-derived cells that express APLNR, we adopted a protocolfor cardiac differentiation, which was stopped at Day 3 when APLNRexpression peaks (Lian et al., 2013). We refer to this intermediary celltype as Day 3-differentiated mesoendoderm. Briefly, Shef4 and HES3 hESCswere dissociated with Accutase, and 0.14 million cells were plated intoeach well of a Matrigel-coated 24 well plate in the presence of 5 μMY-27632 in mTSER1. The media was changed daily for 4 days with mTSER1until the cells became 90% confluent. On Day 0, mTSER1 was then replacedwith differentiation media [RPMI 1640, B-27 supplement with no insulin(Invitrogen)] with 9 μM of GSK3β inhibitor CHIR99201 (BioVision) for 24hours. The cells were further grown for 2 days in RPMI+B27 withoutinsulin until Day 3, when they were used for qPCR, flow cytometry andAP-binding assays.

Example 17 Materials and Methods: Flow Cytometry

Cells were resuspended to 2.5 million/ml and stained with 5 μg/ml of aAPLNR (mAB856, R&D Systems) in PBS/5% FBS/1 mM EDTA for 30 minutes onice, followed by wash in buffer, and stain with a mouse-IgG-AlexaFluo488 (Molecular Probes) for 30 minutes on ice. Flow cytometry wascarried out on LSRII (Becton Dickinson) and data analysis was performedwith FlowJo (Treestar Inc.).

Example 18 Results: ELA is a Conserved Hormone Associated with HumanEmbryonic Pluripotency

Within the human pluripotency circuitry network, which we delineated asthe core intersection of the syn-expression groups (Day et al., 2009;Niehrs and Pollet, 1999) of NANOG, POU5F1 and PRDM14, lies a list of 33transcripts (FIG. 1A), six of which are still unknown oruncharacterized. One, AK092578, was previously reported to be specificto undifferentiated hESCs (Miura et al., 2004). According to UniGene,this transcript is conspicuously expressed in human blastocysts beforeimplantation (FIG. 9A and FIG. 9B). Its expression is dependent on anactive POU5F1 regulatory element 10 kb upstream of its promoter and isdownregulated in POU5F1-depleted hESCs (FIG. 9C and FIG. 9D). Consistentwith this, ELA transcription is highest in undifferentiated hESCs andbecomes rapidly silenced during embryoid body, endodermal, RA-mediatedneuronal and cardiac directed differentiation (FIG. 1B, FIG. 9E to FIG.9H).

Human ELA consists of 3 exons on chromosome 4 that generates atranscript annotated as a non-coding RNA. However, ELA mRNA contains aconserved ORF which encodes a predicted polypeptide of 54 amino acids(FIG. 1C). Phylogenetic analysis revealed that this polypeptide is ahighly conserved protein with a predicted N-terminal signal sequence of22 residues (FIG. 1C). Along with a pair of conserved cysteines, thelast 13 residues are nearly invariant in all vertebrate species (FIG.1C). Based on this prediction, we raised antibodies against the N- andC-termini of the predicted mature ELA peptide (referred hereafter as α Nand α C antibodies) (FIG. 1C). To confirm that ELA is processed forsecretion, human ELA ORF mRNA was microinjected in 4-cell stage Xenopuslaevis embryos. After 10 hours of secretion, we confirmed that ELA wastranslated, processed and secreted by embryos using the α C antibody(FIG. 1D). In the supernatant, processed ELA was of the same size as asynthetically produced recombinant ELA. Increasing amounts of BrefeldinA (BFA), an antibiotic that blocks the exit of secretory proteins fromthe endoplasmic reticulum, was able to block ELA processing andsecretion (FIG. 1D). Unlike the α C antibody, which recognizes bothfull-length and processed ELA, the α N antibody was specific to matureELA, indicating that its epitope is revealed once the signal peptide iscleaved (FIG. 1E). Taken together, these data confirm that ELA belongsto the pluripotency network of hESCs and encodes a potentially solublemature peptide with a molecular weight of less than 4 kDa.

Example 19 Results: ELA is Secreted by hESCs and is Needed for theirSelf-Renewal

ELA is a bona fide endogenous protein because it is detectable byimmunofluorescence using the α N and C antibodies in hESCs and inrelated human embryonic carcinoma cells (hECs) (FIG. 2A and FIG. 10A toFIG. 10C). ELA staining was highest in the Golgi apparatus as evidencedby co-localization with TGN46, a marker of the trans-Golgi network, butis also found in the cytoplasm (FIG. 2A and FIG. 10A). This staining isspecific, as it was markedly reduced upon siRNA- and shRNA-mediated ELAknockdown in both pluripotent cell types (FIG. 2A and FIG. 10C). Weconfirmed that endogenous ELA was indeed secreted because it is readilydetected in the supernatant of cultured hESCs using a sandwich ELISAassay (FIG. 2B). We estimated that over a period of 5 days, solubleendogenous ELA reached nanomolar range concentrations in the supernatantof hESCs (FIG. 2C). To assess ELA function, we next generated stabledoxycycline-inducible shRNA knockdown hESCs against ELA and anon-essential gene β2-MICROGLOBULIN (β2M) (FIG. 10D) (Zafarana et al.,2009). shELA knockdown achieved approximately 85% depletion ofextracellular ELA relative to control levels (FIG. 2C). Prolongeddepletion of ELA, but not β2M, resulted in a gradual loss of hESCscolony morphology and a downregulation of pluripotency markers POU5F1,NANOG, SSEA3 and TRA-1-60 (FIG. 2D, FIG. 10F and FIG. 10G). shELA hESCsinjected into SCID mice did not form teratomas as did control hESCs(FIG. 10E). In line with these results, shELA hESCs displayed markedlyreduced growth rates compared to control and shβ2M hESCs when seeded assingle cells as shown by real-time cell index analysis (FIG. 2E). Thisindex serves a proxy for cell numbers, which is proportional to thesurface area occupied (FIG. 2E). Slower growth rates were alsodocumented in shELA hESC colonies that were on average less than halfthe size of shβ2M hESCs (FIG. 2F). This impairment in cell numbers wasnot due to the activation of G1/S checkpoint or lengthened G0 residence,as evidenced by EDU staining (FIG. 10H). Cell cycle analysis followingrelease from a double thymidine block, which synchronizes hESCs in theG1 phase, confirmed this result (FIG. 2G). Rather, shELA hESCs underwentapoptosis at a significantly higher rate compared to control hESCs, asrevealed by an approximately 40% increase in the number of ANNEXIN V andactivated CASPASE 3-positive cells (FIG. 2H). We conclude from theseobservations that ELA is an endogenous secreted peptide that isessential for the survival and self-renewal of hESCs and that itsdepletion triggers apoptosis and gradual differentiation.

Example 20 Results: Exogenous ELA Promotes Growth and Primes hESCsTowards Mesendoderm Lineages

We next assessed the bioactivity of mature ELA. To this end wesynthetically produced recombinant mature ELA at 98% purity as a 32amino-acid peptide bearing an intramolecular cystine bond betweencysteine residues 39 and 44 (FIG. 3A). This recombinant FITC-labeled ELAwas rapidly up-taken by hESCs (FIG. 3B). We found that mutation of twoinvariant arginines into glycines (R31G and R32G) completely abolishedthe uptake of recombinant ELA (FIGS. 3A and 3B). While shELA hESCsshowed reduced growth, hESCs pulsed with ELA peptide showeddose-dependent enhanced growth relative to untreated cells. This wasindependently documented by cell counts (FIG. 3C), colony size (FIG. 3D)and by real-time measurement of cell indices (FIG. 3E and FIG. 11A). Thedoubly mutated ELA mutant peptide (referred to as ELA^(RR>GG)) waswithout effect in these assays (FIGS. 3D and 3E). Notably, the growth ofshELA hESCs was entirely rescued to normal levels by addition ofrecombinant ELA, but not ELA^(RR>GG), showing that ELA can exert itsrole wholly as a non-cell autonomous factor (FIG. 3F). We thereforehypothesized that its direct inhibition in the extracellular spaceshould yield similar results as its depletion at the mRNA level. Indeed,we found that addition of affinity-purified α C and N antibodies to hESCmedium recapitulated the effects of shELA (FIG. 3G and FIG. 11B),indicating that these antibodies have potent ELA-neutralizing activity.The non-signaling mutant ELA^(RR>GG) peptide was used as a competitiveinhibitor of the α C antibody to prove the specificity of this assay(FIG. 3G). It is noteworthy that recombinant ELA exerted activity onlyon hESCs cultures and no other differentiated cell types including hECs,a human chondrosarcoma cell line or primary human fibroblasts (FIG. 3H).

We observed that in the presence of elevated ELA, following the additionof exogenous ELA peptide to hESC cultures, mesendodermal genes includingGATA6, GATA4, FOXA2, EOMES and BRA were consistently upregulated. Thesesame markers were conversely downregulated in shELA hESCs (FIG. 3I). Inspite of this, hESCs did not appear to lose stemness, as judged by theunchanged levels of cell surface markers SSEA3 and TRA-1-60 (FIG. 3J).This priming towards mesendoderm did not represent a permanent lineagecommitment, since ELA-treated hESCs were not biased towards mesoderm orendoderm lineages during embryoid body differentiation and could equallyexpress NESTIN a neuroectodermal marker (FIG. 3K). Taken together, thesedata suggest that mature ELA functions as an extracellular factor ableto elicit enhanced growth and survival of hESCs. Upon overexpression,ELA poises hESCs towards the mesendoderm lineage but without causingovert lineage commitment.

Example 21 Results: Creating an Allelic Series of Ela Mutants inZebrafish

During zebrafish embryogenesis, ela is expressed from the mid-blastulatransition (MBT) to 3 days post-fertilization (FIGS. 4A and 4B). Withoutany measurable maternal contribution, ela is ubiquitous in dividingcells of the blastoderm before becoming restricted after gastrulation toaxial structures with most prominent expression in the neural tube (FIG.4A). ela is located on chromosome 1 and also consists of 3 exons (FIG.4C). To document the exact function and requirement for ela in vivo, wedesigned and injected custom ZFNs to induce double-stranded breaks inexon 1 of ela, which codes for its signal peptide. Screening of the F1generation allowed us to identify an allelic series (FIG. 4D) ofheterozygous ela fish that were selected and backcrossed at least fivetimes before phenotypic analysis was undertaken.

We analyzed 3 distinct ela alleles. ela^(br21) is a 21 bp deletioncausing a unique 7 amino acid in-frame deletion within the signalpeptide but which leaves the mature peptide intact. ela^(br13) andela^(br15) are frameshift alleles caused by deletion of 13 and 15 bp,respectively, that disrupt the entire mature Ela peptide (FIG. 4D). elanull embryos developed normally up to 50% epiboly, after which migrationanomalies were observed in the germ ring; ela null embryos were easilyscored by eye using the “rough” and constricted appearance of theinvoluting marginal layer at the shield stage (FIG. 4E). At 100%epiboly, when ela expression peaks (FIG. 4B), we confirmed by RT-PCRthat ela^(br13)mRNA was shorter than that of wildtype ela mRNA (FIG.4F), which is consistent with its biallelic genomic deletion. By westernblotting using a C antibodies, the endogenous Ela full-length proteincould be detected in extracts of wt embryos but was absent fromela^(br13) embryos (FIG. 4F).

Example 22 Results: Loss of Ela Causes Embryonic Lethality Due to HeartDysgenesis

All ela homozygous mutant fish showed similar phenotypes and were ofexpected Mendelian recessive ratios (FIG. 5A and FIG. 12A to FIG. 12C),suggesting that the three alleles behave as loss-of-function mutants.ela heterozygous fish were ostensibly normal, while ela null fishpresented with severe cardiac dysplasia ranging from rudimentary heartto no heart (FIG. 5A, FIG. 5B and FIG. 12A to FIG. 12C). Loss, or severereduction of embryonic heart marker cmlc1 was seen in more than 95% ofela null embryos (n>370) regardless of the allele analyzed (FIG. 5C). Noblood circulation was observed and excess erythrocytes accumulated atthe intermediate cell mass (ICM) (FIG. 5A), which was confirmed bysc/upregulation in ela mutant embryos relative to heterozygous siblings(FIG. 5D).

In addition, ela mutant larvae displayed variable posterior truncations,and at times, tailbud duplications. Posterior tissue defects ranged fromloss of ventral fin to complete tail and trunk truncations (FIG. 5A).Unexpectedly, similar phenotypes were also observed upon ela mRNAoverexpression (FIG. 12D). Thousands of ela null embryos from all threegenotypes were obtained and scored into 3 classes according to theseverity of tail defects (FIGS. 5A and 5E). We observed that therelative proportions of each class varied greatly among heterozygouscrosses (FIG. 5E) and even among different clutches of embryos born toidentical parents (data not shown), suggesting notable phenotypicvariance in tail development but not in heart morphogenesis. Remarkably,a very low percentage of ela null mutants develop to fertile adults(FIG. 5F), ruling out any maternal ela effects and permitting homozygouscrosses to yield 100% null clutches. These 3 ela loss-of-functionalleles show that truncation of one third of the Ela signal peptidecauses identical phenotypes as frameshift mutations, indicating that Elarequires an intact signal peptide to be functional in vivo. Moreover,these recessive ela alleles suggest that lowering Ela levels by half isof no obvious consequence whereas its total absence is incompatible toheart development, hematopoiesis and to a lesser extent for tailelongation.

Example 23 Results: ELA is Required for Proper Endoderm Differentiationin Zebrafish and hESCs

In order to understand the embryological origin of the severe cardiacdefects observed in ela mutant fish, we analyzed by whole-mount in situhybridization a series of markers for all three embryonic germ layers.Most prominently, ela mutant gastrulae displayed specific defects in themesendodermal lineage (FIG. 6A-D). Mesoderm, marked by bra, exhibitedimpaired epiboly movements presumably caused by convergent-extensionanomalies. At 100% epiboly ela mutant embryos were delayed and had anopen blastopore with a shorter and thicker notochord relative towildtype (wt) embryos (FIG. 6A). The expression of gata5, which marksmesendoderm and promotes the development of cardiovascular progenitorcells (CPCs), was distinctly altered in ela embryos compared to wtembryos at 75% epiboly; instead of marking a discrete axial populationof cells in the heart-forming region, ela null embryos displayed adistinctive chevron shape around the organizer that was continuous withthe marginal gata5-expressing cells (FIG. 6B). Expression of sox17,which marks definitive endodermal precursors, was equally chevron-shapedin the dorsal side, although sox17 forerunner cells were unaltered bythe absence of ela (FIG. 6C and FIG. 13A). We observed a significantreduction in the total number of sox17⁺ cells at 75% epiboly, which onaverage were reduced by 30 to 40% compared to wt or ela heterozygoussiblings (FIG. 6D).

To test whether human ELA is similarly required for endodermdifferentiation of hESCs, we differentiated hESCs for 5 days and countedthe number of SOX17⁺ definitive endoderm cells. We observed impairedendoderm differentiation of shELA but not shβ2M or control hESCs (FIG.6E, FIG. 13B and FIG. 13C). shELA hESCs showed a significant 45%reduction over control in the number of SOX17⁺ cells which could beentirely rescued by addition of the recombinant ELA peptide (FIG. 6F).We conclude that ELA is essential both in vivo and in vitro forembryonic pluripotency and for the proper differentiation of endodermalprecursors.

Example 24 Results: APLNR is the Cognate Receptor for ELA During HeartDevelopment

Hormonal peptides signal through G-protein coupled Receptors (GPCRs).One such GPCR, the apelin receptor (APLNR), has been implicated in heartdevelopment in fish and mice. The zebrafish mutant grinch, which carriesa recessive W90L missense mutation in aplnrb, and Aplnr knockout miceboth have defects in cardiac morphogenesis (Charo et al., 2009; Scott etal., 2007). Unexpectedly however, loss of APLN, the accepted ligand forAPLNR does not recapitulate the phenotype of grinch in zebrafish orAplnr null mice. We surmised that ELA might be the long sought-afteralternate and earlier ligand for APLNR (Charo et al., 2009; Scott etal., 2007). Unlike most other hormones that have near neutralisoelectric points, ELA and APLN are rich in basic residues and haveisoelectric points above 12 (FIG. 7A), suggesting they might share acommon receptor. For ELA to be the first ligand of APLNR, we contendedthat it should: 1) be expressed concomitantly with aplnr before theonset of gastrulation; 2) be expressed in, or adjacent to,aplnr-expressing cells; 3) phenocopy aplnr mutants and 4) bind to APLNRon the surface of cells. Consistent with previous reports, the onset ofaplnra and aplnrb coincides with that of ela at MBT, whereas aplnexpression begins 5 hours later during gastrulation (FIG. 7B). Cellsexpressing aplnra and aplnrb are in the hypoblast (Zeng et al., 2007),just beneath the overlying enveloping layer where ela is ubiquitouslytranscribed (FIGS. 4A and 7C). We found that the expression of aplnraand aplnrb was also responsive to the loss of ela, displaying a morecondensed pattern at the margin relative to wt embryos (FIG. 7C).Phenotypically, and using an array of markers, we found that aplnrmorphants were indistinguishable from ela null embryos. Both exhibitedpericardial oedema with markedly reduced cmlc1 expression andaccumulation of erythrocytes in the ICM at 30 hours post fertilization(hpf). By six days, all ela mutant and aplnr morphant embryos hadcardiac dysplasia with little to no blood circulation (FIG. 7D-7F).These in vivo data demonstrate that loss of ela phenocopies the loss ofaplnr, arguing that they form a ligand-receptor pair in vivo. Lastly,overexpression of zebrafish aplnra/b and human APLNR in HEK293T cells,which do not express APLNR and do not bind ELA, was sufficient to affordcell-surface binding to ELA conjugated to Alkaline Phosphatase (AP-ELA)(FIG. 7G). In contrast aplnrb^(grinch) was unable to bind AP-ELA norcould GPR15 (FIG. 7H), an orphan GPCR closely related to APLNR(Vassilatis et al., 2003). These results suggest that extracellular ELAcan bind APLNR in a native cellular context. Taken together, our resultsstrongly support the notion that ELA, and not APLN, is the first agonistof APLNR which in tandem direct endodermal differentiation for cardiacdevelopment.

Unexpectedly, APLNR is reported to be absent in hESCs (Vodyanik et al.,2010; Yu et al., 2012), arguing that it is not the ELA receptormediating hESCs self-renewal. Unlike ELA, which is marked by H3K4me3 andactively transcribed in hESCs, APLNR is methylated and not transcribed(FIG. 14A). We confirmed the absence of APLNR transcripts in the Shef4and HES3 hESC lines by qPCR and flow cytometry. In contrast, APLNRtranscripts were upregulated about 2500-fold upon mesendodermdifferentiation, when cell surface APLNR becomes robustly detectable(FIG. 14B and FIG. 14C). Notwithstanding, we performed shRNA-mediateddepletion of APLNR in hESCs to ensure that trace levels of APLNR inhESCs could not account for cell surface binding of ELA. Depletion ofAPLNR in hESCs (FIG. 14B and FIG. 14D) could not reduce binding ofAP-ELA to undifferentiated hESCs, but was sufficient to significantlydiminish the levels of AP-ELA bound to hESC-derived mesendoderm cells(FIG. 14E). These data suggest that while APLNR is both necessary andsufficient to confer cell surface binding to ELA in differentiatedcells, it is not the endogenous receptor for ELA in undifferentiatedhESCs. Consistent with this conclusion, the growth of shAPLNR hESCs wasnot compromised (FIG. 14F), unlike that of shELA hESCs. From theseexperiments, we predict that an alternate ELA receptor exists in hESCsand is responsible for maintaining embryonic self-renewal.

Example 25 Discussion

Our work has uncovered a new peptide hormone with potent embryonicsignaling activity. We surmise that more will surface as carefulexaminations of multi-exonic transcripts are screened for the existenceof phylogenetically conserved small ORFs. Encoded by a transcriptbelieved to be a non-coding RNA, ELABELA is in fact the precursor for asmall secreted peptide found in all vertebrate species. Its mature formELA, is central to self-renewal of hESCs and required for endodermdifferentiation. In vivo, ela specifically affects the mesendodermallineage where its activity, transduced by APLNR, brings about themigration and differentiation of the cardiac lineage. The identity ofELA's receptor in hESCs is unknown and is the subject of intenseinvestigation.

Example 26 Discussion: Regulation of Embryonic Pluripotency by SecretedFactors

A disproportionate emphasis has been placed on transcription factors forthe maintenance of the pluripotency circuit. In recent years, a plethoraof transcription factors has been shown to be critical for thepluripotent state and to enhance reprogramming (Takahashi and Yamanaka,2006). However, no novel secreted factors, along with NODAL/BMP andIGF/FGF that are established factors for pluripotency, have beendiscovered in the last 20 years. Here we present evidence that one suchextracellular molecule, ELA exists and plays an important role instemness. Unlike FGFs which needs to be added exogenously, or secretedby feeder cells to maintain pluripotency in hESCs, ELA is endogenouslysynthesized, secreted and taken up by hESCs in an autocrine and/orparacrine fashion in self-sufficient quantities. The reason why suchmolecules are needed is not entirely clear but the fact that inhibitionof ELA causes rapid apoptosis suggests that it serves as a pro-survivalfactor to counteract the high levels of spontaneous apoptosis that areinherent to hESCs, particularly following dissociation (Wang et al.,2009; Watanabe et al., 2007). In line with this, we note that thedepletion of ELA in hESCs grown as single cells is more detrimental thanhESCs grown in colonies. Alternatively, this might point to theextracellular activity of the peptide, which can be more readilycaptured in a paracrine manner by hESC colonies than it is by singlecells. Also, we note that while endogenous ELA is present inhESC-conditioned media in the nM range, our recombinant peptide,although specific, is bioactive only in the μM range. This findingsuggests existence of possible post-translational modifications onendogenous ELA that are required for its full potency, as is the casefor GHRELIN (Kojima et al., 1999).

Our observation that recombinant ELA is rapidly taken up by hESCs andthat endogenous ELA can be found in the cytoplasm suggests that it has adedicated receptor in these cells. We do not favor the possibility thatELA behaves a self-penetrating peptide despite its very basic amino-acidmake-up (Green and Loewenstein, 1988) because its rapid cellular uptakeis only observed in hESCs and not in other tested cell types. APLNR issilent in hESCs (FIG. 14A to FIG. 14C), we therefore believe thatanother cell surface receptor mediates ELA activity in hESCs.

Example 27 Discussion: The ELA-APLNR Axis in Cardiovascular Development

Stainier and colleagues have shown that although aplnr is required priorto the onset of gastrulation for proper cardiac morphogenesis, its knownligand apln is not expressed until mid-gastrulation (Scott et al.,2007). Similarly in mice, several groups have reported that theknockouts of Apln and Aplnr are not functionally allelic, as would beexpected if a linear and exclusive ligand-receptor relationship linkedApln to Aplnr. This has led some to hypothesize that Aplnr can haveligand-independent functions and recent reports have shown that Aplnrcan respond to stretch in the absence of Apln (Scimia et al., 2012). Analternate and non mutually-exclusive explanation for this phenomenoninvokes the existence of a second ligand for APLNR. Our results stronglysuggest that ELA fulfills this role in early zebrafish embryogenesis,where ela phenocopies the loss of aplnr and directs the migration andamplification of endodermal precursors for proper cardiac ontogeny.

At present, it is unclear if APLN and ELA signal through APLNR to elicitidentical signaling cascades and therefore partly compensate for oneanother. With respect to cardiovascular development however, we notethat apln zebrafish morphants or apln mouse knockouts do not displayovert congenital cardiac anomalies (Kuba et al., 2007; Scott et al.,2007). The ELA-APLNR axis thus appears to be exclusive for cardiacdevelopment and APLN can be insufficient either due to divergingsignaling downstream of APLNR, or simply due to incompatiblespatiotemporal patterns of expression.

How ela in the epibolyzing blastoderm affects the migration anddifferentiation of aplnr-expressing endodermal precursors in thehypoblast is not clear. Our analysis places ela upstream of gata5, oneof the earliest markers of mesendodermal cells, which fails to coalesceat the midline in ela mutants. Mutation in gata5 in faust zebrafishdemonstrates that this transcription factor is required for precardiacmesoderm to migrate to the embryonic midline (Reiter et al., 1999). Themyocardium lineage which is one of the first paraxial cell populationsto migrate into the anterior lateral plate mesoderm (ALPM) is verysensitive to changes in the endoderm lineage, which is specified by theNodal pathway (Schier, 2003). Our loss-of-function alleles placesignaling by Ela upstream, or in parallel to the endodermal-mediatedpathway for heart morphogenesis. Ela can be required for the correctproliferation of endoderm precursor cells as judged by their decreasednumbers in ela mutant fish. Alternatively, or concurrently, Ela canbehave as a chemotactic stimulus that promotes the timely migration ofendodermal cells towards the midline, who in turn guide thecardiovascular progenitor cells (CPCs) towards the APLM and render themcompetent to initiate cardiogenesis.

Example 28 Discussion: Polygamy of Ligand-Receptor Pairing

One receptor, multiple ligands. The TGFβ ligands ACTIVIN and NODAL havedistinct spatiotemporal expression during development which allows forthe same set of receptors to be activated sequentially. In the case ofELA and APLN, likewise, differences in tissue and timing of expressioncould potentially expand the utility of the APLNR signaling cascade.However, unlike ACTIVIN and NODAL which are structurally related TGFβcytokines, ELA and APLN do not share sequence homology besides beingboth very basic proteins. A similar scenario exists within the world ofsmall peptides, where for instance for the Calcitonin-gene-relatedpeptide (CGRP) and adrenomedullin (AM) hormone both signal through thesame GPCR calcitonin-receptor-like receptor (CRLR) depending on whichmember of the single membrane-spanning RAMP protein is present. Byinteracting with RAMP1, CRLR acquires a high affinity for CGRP, whereasby interacting with RAMP2 or RAMP3, CRLR acquires a high affinity for AM(McLatchie et al., 1998). This system enables one receptor to transducethe signals of multiple ligands.

Example 29 Discussion: One Ligand, Multiple Receptors

While ELA signaling appears to be mediated by the APNLR during heartdevelopment, we find that APLNR is not expressed in hESCs where ELA haspotent bioactivity. This would not be unprecedented and examples aboundof ligands signaling through distinct receptors in a context- andtissue-dependent manner. For instance, WNT ligands signal primarilythrough GPCRs of the FRIZZLED family (Niehrs, 2012) but also via RTKsuch as ROR for convergent-extension movements (Hikasa et al., 2002).Likewise, ELA signals through APLNR to direct cardiac progenitormigration in the early embryo whereas it can signal through an as-yetunidentified receptor in hESCs to promote self-renewal and growth. Thisputative “ménage à quatre” suggests the existence of a delicate andfine-tuned relationship between ELA, APLN, APLNR and ELA's hESC-specificreceptor which will warrant the investigation of tissue specificinactivation of single and double ligand and receptor mutants.

Example 30 Discussion: Future Directions

Besides its expression in the pre-implantation human embryo, ELA mRNA isalso found in the adult prostate and kidney in humans. It will beinteresting to examine its role in these exocrine glands as well.Although quite speculative at this stage, several forward-lookingstatements can be made with respect to the possible therapeutic value ofrecombinant ELA. Assuming that ELA signals through APLNR in adults itwill be important to assess if this hormone is endowed with potentcardioprotective and vasodilatory properties as is APLN (Ashley et al.,2005; Maguire et al., 2009) and therefore serve as a novel therapeuticpeptide for cardiac repair/regeneration and blood pressure control. Inthis regard ELA can serve as a potent inducer of cardiac lineages invitro and along this line, potential loss-of-function ELA alleles can beassociated with cardiovascular diseases in the general human population.Coincidentally, since APLNR permits entry of the HIV-1 by serving as aco-receptor (Zou et al., 2000), it will be interesting to determine if,a non-signaling version of ELA can serve as a means to alter andpossibly slow-down AIDS' progression.

Example 31 ELABELA Polypeptide has Cardioprotective Activity

To demonstrate that ELABELA polypeptide confers cardioprotection, we usea mouse or pig model of ischemia and reperfusion injury.

Study Design for Mouse Study

Myocarial ischaemia is induced by 30 minutes left coronary artery (LCA)occlusion and subsequent reperfusion.

Mice are treated with ELABELA polypeptide via the tail vein, 5 minutesbefore reperfusion. Infarct size is assessed the following day (24 hoursafter reperfusion).

Study Design for Pig Study

Thirty female Dalland Landrace pigs (60-70 kg; IDDLO, Lelystad, TheNetherlands), all pretreated with clopidogrel 75 mg/day for 3 days andamiodarone 400 mg/day for 10 days, are randomly assigned to ELABELApolypeptide, non-ELABELA polypeptide, or saline treatment.

The saline group is added to assess a potential effect of fresh,non-conditioned culture medium. In all pigs, MI is induced by 75 minutesof proximal left circumflex coronary artery (LCxCA) ligation and 4 hoursof subsequent reperfusion. An ischemic period of 75 minutes is selectedto inflict severe myocardial injury without inducing completelytransmural myocardial infarction. The 4 hour reperfusion period is used,because infarct size measurement using TTC staining is most reliableafter 3 hours of reperfusion (Birnbaum et al., 1997).

After longer periods of reperfusion, it becomes more difficult to assessoxidative stress status and apoptotic mechanisms. Treatment is initiated5 minutes before the onset of reperfusion by intravenous infusion ofELABELA polypeptide (1.0 ml, 2.0 mg protein) non-ELABELA polypeptide orsaline. Immediately following reperfusion, an additional intracoronarybolus ELABELA polypeptide (4.0 ml, 8.0 mg protein), non-ELABELApolypeptide or saline is given. Myocardial infarct size and function areassessed 4 hours after reperfusion.

MI and Operational Procedure

During the entire operation, ECG, Systemic Arterial Pressure, andcapnogram are monitored continuously. Under general anesthesia asdescribed before (Timmers et al 2007), a median sternotomy is performedand two introduction sheets are inserted in the carotid arteries for a 6Fr guiding catheter and an 8 Fr conductance catheter (CD Leycom,Zoetermeer, the Netherlands).

The distal tip of a Swan Ganz catheter is placed into the pulmonaryartery via the internal jugular vein. Transonic flow probes (TransonicSystems Inc, Ithaca, N.Y.) are placed around the proximal aorta andLCxCA to measure cardiac output and coronary flow, and a wire is placedaround the inferior caval vein to enable functional measurements undervarying loading conditions for PV loops. After functional measurements,10.000 IU of heparin are administered intravenously and sutures aretightened to occlude the proximal LCxCA. Internal defibrillation with 50J is used when ventricular fibrillation occurred. After 75 minutes ofischemia, the LCxCA is reopened by release of the suture. Immediatelyfollowing reperfusion, Nitroglycerine (0.1 mg to prevent no-reflow) isinfused through the LCxCA via the guiding catheter, followed byintracoronary treatment with ELABELA polypeptide, non-ELABELApolypeptide or saline. After 4 hours of reperfusion, the finalfunctional measurements are performed and the heart is explanted forinfarct size analysis.

Mice are anesthetized with Fentanyl (0.05 mg/kg), Dormicum (5 mg/kg) andDomitor (0.5 mg/kg) and intubated using a 24-gauge intravenous catheterwith a blunt end. Mice are artificially ventilated at a rate of 105strokes/min using a rodent ventilator with a mixture of O₂ and N₂O (1:2vol/vol) to which isoflurane (2.5-3.0% vol/vol) is added. The mouse isplaced on a heating pad to maintain the body temperature at 37° C. Thechest is opened in the third intercostal space and an 8-0 prolene sutureis used to occlude the left coronary artery (LCA) for 30 minutes. Thechest is closed and the following day (24 hours later), the hearts areexplanted for infarct size analysis.

Functional Measurements

The ECG, arterial pressure and cardiac output, are digitized at asampling rate of 250 Hz and stored for offline analysis (Leycom CFL-512,CD Leycom). Left ventricular (LV) pressure and volume are measured usingthe conductance catheter method, as described previously (Timmers et al2007). LV pressure and volume signals derived from the conductancecatheter are displayed and acquired at a 250-Hz sampling rate with aLeycom CFL-512 (CD Leycom).

Data are acquired during steady state and during temporal caval veinocclusion, all with the ventilator turned off at end expiration.Analysis of the pressure-volume loops is performed with custom softwareas described previously (Steendijk et al 1998). In addition, short-axisepicardial ultrasound images (Prosound SSD-5000, 5-MHz probe UST-5280-5,Aloka Holding Europe AG, Zug, Switzerland) are obtained at themidpapillary muscle level. Wall thickness (WT) of the infarct area,remote area (septum) and LV internal area (LVia) are measured at enddiastole (ED) and end systole (ES).

Systolic wall thickening (SWT) is calculated as[(WT(ES)−WT(ED))/WT(ED)]*100%, fractional area shortening (FAS) as[(LVia(ES)−LVia (ED))/LVia (ED)]*100%, and left ventricular ejectionfraction (LVEF) as [(EDV−ESV)/EDV]*100%. The end-diastolic chamberstiffness is quantified by means of linear regression of theend-diastolic pressure-volume relationship. Echocardiography and PVloops are measured before MI, 1 hour after ischemia and 4 hours afterreperfusion. To challenge stunned myocardium, additional measurementsare performed during pharmaceutically induced stress by intravenousdobutamine infusion (2.5 μg/kg/min and 5.0 μg/kg/min).

Infarct Size

Just prior to excision of the heart, the LCxCA (pigs) or LCA (mice) isreligated at exactly the same spot as for the induction of the MI. Evansblue dye is infused through the coronary system to delineate the area atrisk (AAR). The heart is then excised, the LV is isolated and cut into 5slices from apex to base.

The slices are incubated in 1% triphenyltetrazolium chloride (TTC,Sigma-Aldrich Chemicals, Zwijndrecht, the Netherlands) in 37° C.Sorensen buffer (13.6 g/L KH₂PO₄+17.8 g/L Na₂H PO₄ 2H₂O, pH 7.4) for 15minutes to discriminate infarct tissue from viable myocardium.

All slices are scanned from both sides, and in each slide, the infarctarea is compared with area at risk and the total area by use of digitalplanimetry software (Image J). After correction for the weight of theslices, infarct size is calculated as a percentage of the AAR and of theLV.

Example 32 ELABELA Polypeptide Protects Against MyocardialIschaemia-Reperfusion Injury

It is known that the APLN/APLNR (APELIN/APELIN Receptor) axis hasprotective effects in myocardial ischemia-reperfusion injury.

Accordingly, ELABELA constitutes an important therapeutic option formyocardial ischaemia-reperfusion injury.

Materials and Methods

The following publications describe experiments showing the role of apeptide hormone (APELIN) in protection against myocardialischaemia-reperfusion injury:

Tao J, ZhuW, Li Y, et al. Apelin-13 protects the heart againstischemia-reperfusion injury through inhibition of ER-dependent apoptoticpathways in a time-dependent fashion. Am J Physiol Heart Circ Physiol2011; 301:H1471-86.

Zeng X J, Zhang L K, Wang H X, Lu L Q, Ma L Q, Tang C S. Apelin protectsheart against ischemia/reperfusion injury in rat. Peptides 2009;30:1144-52.

Simpkin J C, Yellon D M, Davidson S M, Lim S Y, Wynne A M, Smith C C.Apelin-13 and apelin-36 exhibit direct cardioprotective activity againstischemia-reperfusion injury. Basic Res Cardiol 2007; 102:518-28.

The experiments described in the above publications are repeated, withthe replacement of APELIN with ELABELA.

Results

When the above experiments are carried out, ELABELA administered duringreperfusion is seen to significantly decrease infarct size in subjectshearts.

Without wishing to be bound by theory, we believe that administration ofELABELA protect hearts against ischemia-reperfusion injury throughactivation of PI3K/Akt and ERK pathways and/or can inhibit generation ofreactive oxygen species.

ELABELA can therefore be used for treating, preventing or alleviatingmyocardial ischaemia reperfusion injury in an individual.

Example 33 ELABELA Polypeptide Protects Against Coronary Artery DiseaseMaterials and Methods

The following publications describe experiments showing the role of apeptide hormone (APELIN) in protection against coronary artery disease:

Azizi Y, Faghihi M, Imani A, Roghani M, Nazari A. Post-infarct treatmentwith [Pyr1]-apelin-13 reduces myocardial damage through reduction ofoxidative injury and nitric oxide enhancement in the rat model ofmyocardial infarction. Peptides 2013; 46:76-82.

Li L, Zeng H, Chen J X. Apelin-13 increases myocardial progenitor cellsand improves repair postmyocardial infarction. Am J Physiol Heart CircPhysiol 2012; 303:H605-18.

Pisarenko O I, Serebryakova L I, Pelogeykina Y A, et al. In vivoreduction of reperfusion injury to the heart with apelin-12 peptide inrats. Bull Exp Biol Med 2011; 152:79-82.

The experiments described in the above publications are repeated, withthe replacement of APELIN with ELABELA.

Results

When the above experiments are carried out, injection of ELABELA tosubjects is found to limit the myocardial infarction size and reducedamage to cardiomyocytes.

Without wishing to be bound by theory, we believe that the effects ofELABELA are achieved by significantly attenuating myocardial damagethrough the reduction of oxidative injury and enhancement of NitricOxide (NO) production.

In addition, ELABELA is found to promote angiogenesis and amelioratecardiac repair postmyocardial infarction.

ELABELA can therefore be used for treating, preventing or alleviatingcoronary artery disease in an individual.

Example 34 ELABELA Polypeptide Protects Against ArtherosclerosisMaterials and Methods

The following publication describes experiments showing the role of apeptide hormone (APELIN) in protection against artherosclerosis:

Chun H J, Ali Z A, Kojima Y, et al. Apelin signaling antagonizes Ang IIeffects in mouse models of atherosclerosis. J Clin Invest 2008;118:3343-54.

The experiments described in the above publication are repeated, withthe replacement of APELIN with ELABELA.

Results

When the above experiments are carried out, we find that, like APLN,ELABELA can protect against artherosclerosis. We find that ELABELAantagonizes the vascular disease-promoting actions of Ang II andmitigates the Ang II-mediated increase in atherosclerosis burden. Italso is seen to inhibit abdominal aortic aneurysm formation and rupture.

ELABELA can therefore be used for treating, preventing or alleviatingartheroscloersis in an individual.

Example 35 ELABELA Polypeptide Protects Against Heart Failure

According to the methods and compositions described here, ELABELAexhibits beneficial effects in the cardiovascular system and/or improvescardiac repair.

Accordingly, ELABELA can be used as, or as part of, therapeutic regimensto treat patients with heart failure.

Materials and Methods

The following publications describe experiments showing the role of apeptide hormone (APELIN) in protection against heart failure:

Scimia M C, Hurtado C, Ray S, Metzler S, Wei K, Wang J, Woods C E,Purcell N H, Catalucci D, Akasaka T, Bueno O F, Vlasuk G P, Kaliman P,Bodmer R, Smith L H, Ashley E, Mercola M, Brown J H, Ruiz-Lozano P.(2012), APJ acts as a dual receptor in cardiac hypertrophy. Nature 2012Aug. 16; 488(7411):394-8.

Sato T, Suzuki T, Watanabe H, Kadowaki A, Fukamizu A, Liu P P, Kimura A,Ito H, Penninger J M, Imai Y, Kuba K. (2013) Apelin is a positiveregulator of ACE2 in failing hearts. J Clin Invest. 2013 Nov. 1.

The experiments described in the above publications are repeated, withthe replacement of APELIN with ELABELA.

Results

When the above experiments are carried out, we find that, like APLN,ELABELA is able to provide protection against heart failure.

Without wishing to be bound by theory, we believe that ELABELAcrosstalks with the renin-angiotensin system via theAngiotensin-converting enzyme 2 (ACE2)/Ang II/Ang 1-7 axis to increasecardiac contractility and control heart failure.

Dysregulation of the ELA/APLNR system can therefore be involved in thepredisposition to cardiovascular diseases. Accordingly, we propose thatenhancing ELABELA action has important therapeutic effects.

Furthermore administration of ELABELA can be used to blunt cardiachypertrophy as does APELIN following sustained pressure overload bytransaortic constriction (TAC), a model for heart failure.

ELABELA can therefore be used for treating, preventing or alleviatingheart failure in an individual.

Example 36 ELABELA Polypeptide Protects Against Hypertension

Like APELIN, we propose that ELABELA acts as a vasodilator andeffectively lowers blood pressure. Accordingly, ELABELA can be used as,or as part of, therapeutic regimens to treat patients with hypertension.

Materials and Methods

The following publications describe experiments showing the role of apeptide hormone (APELIN) in protection against hypertension:

Tatemoto K, Takayama K, Zou M X, et al. The novel peptide apelin lowersblood pressure via a nitric oxide-dependent mechanism. RegulatoryPeptides. 2001; 99(2-3):87-92.

Cheng X, Cheng X S, Pang C C. Venous dilator effect of apelin, anendogenous peptide ligand for the orphan APJ receptor, in consciousrats. European Journal of Pharmacology. 2003; 470(3):171-175.

Japp A G, Cruden N L, Amer D A, et al. Vascular effects of apelin invivo in man. Journal of the American College of Cardiology. 2008;52(11):908-913.

The experiments described in the above publications are repeated, withthe replacement of APELIN with ELABELA.

Results

When the experiments described above are carried out, it is found thatadministration of ELABELA causes a reduction in blood pressure. Withoutwishing to be bound by theory, we believe that it does so by functioningas an arterial and/or venous dilator via the nitric oxide-dependentpathway.

ELABELA can therefore be used for treating, preventing or alleviatinghypertension in an individual.

Example 37 ELABELA Polypeptide Protects Against HIV Infection and AIDS

The identification of specific inhibitors of APLNR-mediated HIV-1 entrywould greatly assist efforts to find new reagents to block HIV-1infection.

Materials and Methods

The following publications describe experiments showing the role of apeptide hormone (APELIN) in protection against HIV infection and AIDS:

Cayabyab M, Hinuma S, Farzan M, Choe H, Fukusumi S, Kitada C, NishizawaN, Hosoya M, Nishimura O, Messele T, Pollakis G, Goudsmit J, Fujino M,Sodroski J. (2000) Apelin, the natural ligand of the orphanseven-transmembrane receptor APJ, inhibits human immunodeficiency virustype 1 entry. J Virol. December; 74(24): 11972-6.

Xuejun Fan, Naiming Zhou, Xiaoling Zhang, Muhammad Mukhtar, Zhixian Lu,Jianhua Fang, Garrett C. DuBois, and Roger J. Pomerantz. (2003)Structural and Functional Study of the Apelin-13 Peptide, an EndogenousLigand of the HIV-1 Coreceptor, API Biochemistry 42, 10163-10168 10163.

The experiments described in the above publications are repeated, withthe replacement of APELIN with ELABELA.

Results

When the above experiments are performed, it is found that ELABELAprevents or slows-down HIV-1 entry.

ELABELA can be particularly useful for central nervous system infectionin patients with HIV-1-induced dementia given that APLNR is widelyexpressed in the brain.

ELABELA can therefore be used for treating, preventing or alleviatingHIV infection or AIDS, or both, in an individual.

Example 38 ELABELA Polypeptide Protects Against Pulmonary ArterialHypertension

The APLN/APLNR system plays a key role in the occurrence and developmentof cardiovascular diseases. Targeting the ELA/APLNR axis also representsa new class of potential therapeutic agents for pulmonary arterialhypertension (PAH).

Materials and Methods

The following publications describe experiments showing the role of apeptide hormone (APELIN) in protection against pulmonary arterialhypertension:

Alastalo T P, Li M, Perez Vde J, et al. Disruption ofPPARgamma/beta-catenin-mediated regulation of apelin impairs BMP-inducedmouse and human pulmonary arterial EC survival. J Clin Invest 2011;121:3735-46.

Falcao-Pires I, Goncalves N, Henriques-Coelho T, Moreira-Goncalves D,Roncon Albuquerque Jr R, Leite-Moreira A F. Apelin decreases myocardialinjury and improves right ventricular function in monocrotaline-inducedpulmonary hypertension. Am J Physiol Heart Circ Physiol 2009;296:H2007-14.

The experiments described in the above publications are repeated, withthe replacement of APELIN with ELABELA.

Results

When the above experiments are performed, it is found that ELABELAalleviates symptoms of pulmonary arterial hypertension.

ELABELA can therefore be used for treating, preventing or alleviatingpulmonary arterial hypertension in an individual.

Example 39 ELABELA Polypeptide Treats Erectile Dysfunction Materials andMethods

The following publication describes experiments showing the role of apeptide hormone (APELIN) in treating erectile dysfunction:

Kwon M H, Tuvshintur B, Kim W J, Jin H R, Yin G N, Song K M, Choi M J,Kwon K D, Batbold D, Ryu J K, Suh J K. Expression of the Apelin-APJPathway and Effects on Erectile Function in a Mouse Model ofVasculogenic Erectile Dysfunction. J Sex Med. 2013 Apr. 11. doi:10.1111/jsm.12158.

The experiments described in the above publications are repeated, withthe replacement of APELIN with ELABELA.

Results

When the above experiments are performed, it is found that ELABELAprovides a significant restoration of erectile function.

ELABELA can therefore be used for treating, preventing or alleviatingerectile dysfunction in an individual.

REFERENCES

-   Ashley, E. A., Powers, J., Chen, M., Kundu, R., Finsterbach, T.,    Caffarelli, A., Deng, A., Eichhorn, J., Mahajan, R., Agrawal, R., et    al. (2005). The endogenous peptide apelin potently improves cardiac    contractility and reduces cardiac loading in vivo. Cardiovascular    research 65, 73-82.-   Bonecchi, R., Galliera, E., Borroni, E. M., Corsi, M. M., Locati,    M., and Mantovani, A. (2009). Chemokines and chemokine receptors: an    overview. Front Biosci (Landmark Ed) 14, 540-551.-   Cederlund, A., Gudmundsson, G. H., and Agerberth, B. (2011).    Antimicrobial peptides important in innate immunity. FEBS J 278,    3942-3951.-   Charo, D. N., Ho, M., Fajardo, G., Kawana, M., Kundu, R. K.,    Sheikh, A. Y., Finsterbach, T. P., Leeper, N. J., Ernst, K. V.,    Chen, M. M., et al. (2009). Endogenous regulation of cardiovascular    function by apelin-APJ. Am J Physiol Heart Circ Physiol 297,    H1904-1913.-   Cummings, D. E., Clement, K., Purnell, J. Q., Vaisse, C., Foster, K.    E., Frayo, R. S., Schwartz, M. W., Basdevant, A., and Weigle, D. S.    (2002). Elevated plasma ghrelin levels in Prader Willi syndrome. Nat    Med 8, 643-644.-   D'Aniello, C., Lonardo, E., Iaconis, S., Guardiola, O., Liguoro, A.    M., Liguori, G. L., Autiero, M., Carmeliet, P., and Minchiotti, G.    (2009). G protein-coupled receptor APJ and its ligand apelin act    downstream of Cripto to specify embryonic stem cells toward the    cardiac lineage through extracellular signal-regulated kinase/p70S6    kinase signaling pathway. Circulation research 105, 231-238.-   Dalton, S. (2013). Signaling networks in human pluripotent stem    cells. Curr Opin Cell Biol 25, 241-246.-   Day, A., Dong, J., Funari, V. A., Harry, B., Strom, S. P., Cohn, D.    H., and Nelson, S. F. (2009). Disease gene characterization through    large-scale co-expression analysis. PloS one 4, e8491.-   Frith, M. C., Forrest, A. R., Nourbakhsh, E., Pang, K. C., Kai, C.,    Kawai, J., Carninci, P., Hayashizaki, Y., Bailey, T. L., and    Grimmond, S. M. (2006). The abundance of short proteins in the    mammalian proteome. PLoS Genet 2, e52.-   Green, M., and Loewenstein, P. M. (1988). Autonomous functional    domains of chemically synthesized human immunodeficiency virus tat    trans-activator protein. Cell 55, 1179-1188.-   Hikasa, H., Shibata, M., Hiratani, I., and Taira, M. (2002). The    Xenopus receptor tyrosine kinase Xror2 modulates morphogenetic    movements of the axial mesoderm and neuroectoderm via Wnt signaling.    Development 129, 5227-5239.-   Hughes, C. S., Nuhn, A. A., Postovit, L. M., and Lajoie, G. A.    (2011). Proteomics of human embryonic stem cells. Proteomics 11,    675-690.-   Inniss, K., and Moore, H. (2006). Mediation of apoptosis and    proliferation of human embryonic stem cells by    sphingosine-1-phosphate. Stem cells and development 15, 789-796.-   Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., and    Kangawa, K. (1999). Ghrelin is a growth-hormone-releasing acylated    peptide from stomach. Nature 402, 656-660.-   Kuba, K., Zhang, L., Imai, Y., Arab, S., Chen, M., Maekawa, Y.,    Leschnik, M., Leibbrandt, A., Markovic, M., Schwaighofer, J., et al.    (2007). Impaired heart contractility in Apelin gene-deficient mice    associated with aging and pressure overload. Circulation research    101, e32-42.-   Link, V., Shevchenko, A., and Heisenberg, C. P. (2006). Proteomics    of early zebrafish embryos. BMC Dev Biol 6, 1.-   Maguire, J. J., Kleinz, M. J., Pitkin, S. L., and Davenport, A. P.    (2009). [Pyr1]apelin-13 identified as the predominant apelin isoform    in the human heart: vasoactive mechanisms and inotropic action in    disease. Hypertension 54, 598-604.-   McLatchie, L. M., Fraser, N. J., Main, M. J., Wise, A., Brown, J.,    Thompson, N., Solari, R., Lee, M. G., and Foord, S. M. (1998). RAMPs    regulate the transport and ligand specificity of the    calcitonin-receptor-like receptor. Nature 393, 333-339.-   Miura, T., Luo, Y., Khrebtukova, I., Brandenberger, R., Zhou, D.,    Thies, R. S., Vasicek, T., Young, H., Lebkowski, J., Carpenter, M.    K., et al. (2004). Monitoring early differentiation events in human    embryonic stem cells by massively parallel signature sequencing and    expressed sequence tag scan. Stem Cells Dev 13, 694-715.-   Montague, C. T., Farooqi, I. S., Whitehead, J. P., Soos, M. A., Rau,    H., Wareham, N. J., Sewter, C. P., Digby, J. E., Mohammed, S. N.,    Hurst, J. A., et al. (1997). Congenital leptin deficiency is    associated with severe early-onset obesity in humans. Nature 387,    903-908.-   Niehrs, C. (2012). The complex world of WNT receptor signalling.    Nature reviews Molecular cell biology 13, 767-779.-   Niehrs, C., and Pollet, N. (1999). Synexpression groups in    eukaryotes. Nature 402, 483-487.-   Nishino, S., Ripley, B., Overeem, S., Lammers, G. J., and Mignot, E.    (2000). Hypocretin (orexin) deficiency in human narcolepsy. Lancet    355, 39-40.-   Peyron, C., Faraco, J., Rogers, W., Ripley, B., Overeem, S.,    Charnay, Y., Nevsimalova, S., Aldrich, M., Reynolds, D., Albin, R.,    et al. (2000). A mutation in a case of early onset narcolepsy and a    generalized absence of hypocretin peptides in human narcoleptic    brains. Nat Med 6, 991-997.-   Rasmussen, S. G., Choi, H. J., Rosenbaum, D. M., Kobilka, T. S.,    Thian, F. S., Edwards, P. C., Burghammer, M., Ratnala, V. R.,    Sanishvili, R., Fischetti, R. F., et al. (2007). Crystal structure    of the human beta2 adrenergic G-protein-coupled receptor. Nature    450, 383-387.-   Reiter, J. F., Alexander, J., Rodaway, A., Yelon, D., Patient, R.,    Holder, N., and Stainier, D. Y. (1999). Gata5 is required for the    development of the heart and endoderm in zebrafish. Genes Dev 13,    2983-2995.-   Reversade, B., and De Robertis, E. M. (2005). Regulation of ADMP and    BMP2/4/7 at opposite embryonic poles generates a self-regulating    morphogenetic field. Cell 123, 1147-1160.-   Schier, A. F. (2003). Nodal signaling in vertebrate development.    Annu Rev Cell Dev Biol 19, 589-621.-   Scimia, M. C., Hurtado, C., Ray, S., Metzler, S., Wei, K., Wang, J.,    Woods, C. E., Purcell, N. H., Catalucci, D., Akasaka, T., et al.    (2012). APJ acts as a dual receptor in cardiac hypertrophy. Nature    488, 394-398.-   Scott, I. C., Masri, B., D'Amico, L. A., Jin, S. W., Jungblut, B.,    Wehman, A. M., Baier, H., Audigier, Y., and Stainier, D. Y. (2007).    The g protein-coupled receptor agtrl1b regulates early development    of myocardial progenitors. Developmental cell 12, 403-413.-   Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent    stem cells from mouse embryonic and adult fibroblast cultures by    defined factors. Cell 126, 663-676.-   van den Pol, A. N. (2012). Neuropeptide transmission in brain    circuits. Neuron 76, 98-115.-   Vassilatis, D. K., Hohmann, J. G., Zeng, H., Li, F., Ranchalis, J.    E., Mortrud, M. T., Brown, A., Rodriguez, S. S., Weller, J. R.,    Wright, A. C., et al. (2003). The G protein-coupled receptor    repertoires of human and mouse. Proc Natl Acad Sci USA 100,    4903-4908.-   Vodyanik, M. A., Yu, J., Zhang, X., Tian, S., Stewart, R.,    Thomson, J. A., and Slukvin, II (2010). A mesoderm-derived precursor    for mesenchymal stem and endothelial cells. Cell Stem Cell 7,    718-729.-   Wang, X., Lin, G., Martins-Taylor, K., Zeng, H., and    Xu, R. H. (2009) Inhibition of caspase-mediated anoikis is critical    for basic fibroblast growth factor-sustained culture of human    pluripotent stem cells. The Journal of biological chemistry 284,    34054-34064.-   Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M.,    Wataya, T., Takahashi, J. B., Nishikawa, S., Muguruma, K., and    Sasai, Y. (2007). A ROCK inhibitor permits survival of dissociated    human embryonic stem cells. Nat Biotechnol 25, 681-686.-   Yu, Q. C., Hirst, C. E., Costa, M., Ng, E. S., Schiesser, J. V.,    Gertow, K., Stanley, E. G., and Elefanty, A. G. (2012). APELIN    promotes hematopoiesis from human embryonic stem cells. Blood 119,    6243-6254.-   Zafarana, G., Avery, S. R., Avery, K., Moore, H. D., and    Andrews, P. W. (2009). Specific knockdown of OCT4 in human embryonic    stem cells by inducible short hairpin RNA interference. Stem Cells    27, 776-782.-   Zeng, X. X., Wilm, T. P., Sepich, D. S., and Solnica-Krezel, L.    (2007). Apelin and its receptor control heart field formation during    zebrafish gastrulation. Developmental cell 12, 391-402.-   Zou, M. X., Liu, H. Y., Haraguchi, Y., Soda, Y., Tatemoto, K., and    Hoshino, H. (2000). Apelin peptides block the entry of human    immunodeficiency virus (HIV). FEBS letters 473, 15-18.-   Alexander, J., and Stainier, D. Y. (1999). A molecular pathway    leading to endoderm formation in zebrafish. Current biology: CB 9,    1147-1157.-   Lian, X., Zhang, J., Azarin, S. M., Zhu, K., Hazeltine, L. B., Bao,    X., Hsiao, C., Kamp, T. J., and Palecek, S. P. (2013). Directed    cardiomyocyte differentiation from human pluripotent stem cells by    modulating Wnt/beta-catenin signaling under fully defined    conditions. Nature protocols 8, 162-175.-   Matys, V., Kel-Margoulis, O. V., Fricke, E., Liebich, I., Land, S.,    Barre-Dirrie, A., Reuter, I., Chekmenev, D., Krull, M., Hornischer,    K., et al. (2006). TRANSFAC and its module TRANSCompel:    transcriptional gene regulation in eukaryotes. Nucleic acids    research 34, D108-110.-   Schoenebeck, J. J., and Yelon, D. (2007). Illuminating cardiac    development: Advances in imaging add new dimensions to the utility    of zebrafish genetics. Semin Cell Dev Biol 18, 27-35.-   Schulte-Merker, S., van Eeden, F. J., Halpern, M. E., Kimmel, C. B.,    and Nusslein-Volhard, C. (1994). no tail (ntl) is the zebrafish    homologue of the mouse T (Brachyury) gene. Development 120,    1009-1015.-   Tiscornia, G., Singer, O., and Verma, I. M. (2006a). Design and    cloning of lentiviral vectors expressing small interfering RNAs.    Nature protocols 1, 234-240.-   Tiscornia, G., Singer, O., and Verma, I. M. (2006b). Production and    purification of lentiviral vectors. Nature protocols 1, 241-245.-   Birnbaum Y, Hale S L, Kloner R A. Differences in reperfusion length    following 30 minutes of ischemia in the rabbit influence infarct    size, as measured by triphenyltetrazolium chloride staining. J Mol    Cell Cardiol. 1997; 29(2):657-666.-   Timmers L, Sluijter J P, Verlaan C W, Steendijk P, Cramer M J, Emons    M, Strijder C, Grundeman P F, Sze S K, Hua L, Piek J J, Borst C,    Pasterkamp G, de Kleijn D P. Cyclooxygenase-2 inhibition increases    mortality, enhances left ventricular remodeling, and impairs    systolic function after myocardial infarction in the pig.    Circulation. 2007; 115(3):326-332.-   Steendijk P, Baan J, Jr., Van der Velde E T, Baan J. Effects of    critical coronary stenosis on global systolic left ventricular    function quantified by pressure-volume relations during dobutamine    stress in the canine heart. J Am Coll Cardiol. 1998; 32(3):816-826.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”.

Each of the applications and patents mentioned in this document, andeach document cited or referenced in each of the above applications andpatents, including during the prosecution of each of the applicationsand patents (“application cited documents”) and any manufacturer'sinstructions or catalogues for any products cited or mentioned in eachof the applications and patents and in any of the application citeddocuments, are hereby incorporated herein by reference. Furthermore, alldocuments cited in this text, and all documents cited or referenced indocuments cited in this text, and any manufacturer's instructions orcatalogues for any products cited or mentioned in this text, are herebyincorporated herein by reference.

Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the claims.

SEQUENCE LISTINGS ELABELA polypeptide signature sequence SEQ ID NO: 1CXXXRCXXXHSRVPFP Homo ELABELA mature polypeptide SEQ ID NO: 2QRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP Peromyscus ELABELA mature polypeptideSEQ ID NO: 3 QRPVNFPKKRKVYRHNCFRRRCVPLHSRVPFPRattus ELABELA mature polypeptide SEQ ID NO: 4EKSVNFPRRRKLYRHNCFRRRCISLHSRVPFP Mus ELABELA mature polypeptideSEQ ID NO: 5 QKPVNFPRRRKLYRHNCFRRRCIPLHSRVPFPBos ELABELA mature polypeptide SEQ ID NO: 6QRQANLAMRRKLHRHNCLQRRCMPLHSRVPFP Sus ELABELA mature polypeptideSEQ ID NO: 7 QRPANLAVRRKLHRHNCLQRRCMPLHSRVPFPDasypsu ELABELA mature polypeptide SEQ ID NO: 8QRPANLAMRRKLHRHNCFQRRCMPLHSRVPFP Trichosurus ELABELA mature polypeptideSEQ ID NO: 9 QRPGNLALRRKPHRHICPQRRCMPLHSRVPFPGallus ELABELA mature polypeptide SEQ ID NO: 10QRPANLALRRKLHRHNCSHRRCMPLHSRVPFP Gekko ELABELA mature polypeptideSEQ ID NO: 11 QRPANLSLRRKLHRQHCSHRRCMPLHSRVPFPAnolis ELABELA mmature polypeptide SEQ ID NO: 12QRPANLASRRKLHRHHCSHRRCMPLHSRVPFP Xenopus ELABELA mature polypeptideSEQ ID NO: 13 QKPANLAQRRRIHRHNCFLKRCIPLHSRVPFPAmbystoma ELABELA mature polypeptide SEQ ID NO: 14QRPVNAAHRRRLHRHNCSLRRCMPLHSRVPFP Oryzias ELABELA maature polypeptideSEQ ID NO: 15 ARPDFLNLRRKYHRHHCLHRRCMPLHSRVPFPCallorhinchus ELABELA mature polypeptide SEQ ID NO: 16QKSGNSWRRKKMQRRNCWHRRCLPFHSRVPFP Oncorhynchus ELABELA mature polypeptideSEQ ID NO: 17 VRPDILNIRRRYHRHHCPHRRCMPLHSRVPFPDanio ELABELA mature polypeptide SEQ ID NO: 18DKHGTKHDFLNLRRKYRRHNCPKKRCLPLHSRVPFP Human ELABELA signal sequenceSEQ ID NO: 19 MRFQQFLFAFFIFIMSLLLISGHomo ELABELA polypeptide with signal sequence (bold) SEQ ID NO: 20MRFQQFLFAFFIFIMSLLLISGQRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFPPeromyscus ELABELA polypeptide with signal sequence (bold) SEQ ID NO: 21MRFQHYFLVFFIFAMSLLFITEQRPVNFPKKRKVYRHNCFRRRCVPLHSRVPFPRattus ELABELA polypeptide with signal sequence (bold) SEQ ID NO: 22MRFQPLFWVFFIFAMSLLFITEEKSVNFPRRRKLYRHNCFRRRCISLHSRVPFPMus ELABELA polypeptide with signal sequence (bold) SEQ ID NO: 23MRFQPLFWVFFIFAMSLLFISEQKPVNFPRRRKLYRHNCFRRRCIPLHSRVPFPBos ELABELA polypeptide with signal sequence (bold) SEQ ID NO: 24MRFHQFFLLFVIFMLSLLLIHGQRQANLAMRRKLHRHNCLQRRCMPLHSRVPFPSus ELABELA polypeptide with signal sequence (bold) SEQ ID NO: 25MRFRQFFLVFFIFMMNLLLICGQRPANLAVRRKLHRHNCLQRRCMPLHSRVPFPDasypus ELABELA polypeptide with signal sequence (bold) SEQ ID NO: 26MKFQQFFYVFFVFIMSLLLINGQRPANLAMRRKLHRHNCFQRRCMPLHSRVPFPTrichosurus ELABELA polypeptide with signal sequence (bold)SEQ ID NO: 27 MRFQLLFFLFLFFTMGILLIDGQRPGNLALRRKPHRHICPQRRCMPLHSRVPFPGallus ELABELA polypeptide with signal sequence (bold) SEQ ID NO: 28MRLRRLLCVVFLLLVSLLPAAAQRPANLALRRKLHRHNCSHRRCMPLHSRVPFPGekko ELABELA polypeptide with signal sequence (bold) SEQ ID NO: 29MRLQLLLLTCFLILTGVLLGNGQRPANLSLRRKLHRQHCSHRRCMPLHSRVPFPAnolis ELABELA polypeptide with signal sequence (bold) SEQ ID NO: 30MRLQQLLLTWFLLLAGALLINGQRPANLASRRKLHRHHCSHRRCMPLHSRVPFPXenopus ELABELA polypeptide with signal sequence (bold) SEQ ID NO: 31MDFQKLLYALFFILMSLLLINGQKPANLAQRRRIHRHNCFLKRCIPLHSRVPFPAmbystoma ELABELA polypeptide with signal sequence (bold) SEQ ID NO: 32MKWQKLLAILFWILMGALLVNGQRPVNAAHRRRLHRHNCSLRRCMPLHSRVPFPOryzias ELABELA polypeptide with signal sequence (bold) SEQ ID NO: 33MRVWNLLYLLLLLAAALAPVFSARPDFLNLRRKYHRHHCLHRRCMPLHSRVPFPCallorhinchus ELABELA polypeptide with signal sequence (bold)SEQ ID NO: 34 MRFQHLLHIILLLCTSLLLISGQKSGNSWRRKKMQRRNCWHRRCLPFHSRVPFPOncorhynchus ELABELA polypeptide with signal sequence (bold)SEQ ID NO: 35 MRIISLLYLLLLVTVLGSVSSVRPDILNIRRRYHRHHCPHRRCMPLHSRVPFPDanio ELABELA polypeptide with signal sequence (bold) SEQ ID NO: 36MRFFHPLYLLLLLLTVLVLISADKHGTKHDFLNLRRKYRRHNCPKKRCLPLHSRVPFPHuman (Homo sapiens) ELABELA cDNA sequence SEQ ID NO: 37ATGAGATTTCAGCAATTCCTTTTTGCATTTTTTATTTTTATTATGAGTCTTCTCCTTATCAGCGGACAGAGACCAGTTAATTTGACCATGAGAAGAAAACTGCGCAAACACAATTGCCTTCAGAGGAGATGTATGCCTCTCCATTCACGAGTACCCTTCCCCTGAMouse (Mus musculus) ELABELA cDNA sequence SEQ ID NO: 38ATGCGATTCCAGCCCCTTTTTTGGGTATTTTTTATTTTTGCCATGAGTCTCCTTTTTATCAGTGAACAGAAACGAGTTAACTTTCCCAGGAGAAGAAAACTATACAGACACAACTGCTTTCGCAGGAGATGCATTCCACTTCATTCTCGAGTGCCCTTCCCATGAChicken (Gallus gallus) ELABELA cDNA sequence SEQ ID NO: 39ATGAGGCTCCGGCGGCTGCTGTGCGTCGTGTTCCTGCTCCTGGTCAGCCTGCTACCTGCCGCCGCGCAGAGACCGGCCAACCTGGCCCTGCGCAGGAAGCTGCACCGACACAACTGCTCGCACCGGCGGTGCATGCCGCTCCACTCCCGCGTGCCCTTTCCCTGAXenopus (Xenopus laevis) ELABELA cDNA sequence SEQ ID NO: 40ATGGATTTTCAGAAATTATTGTATGCCTTATTTTTCATTCTGATGAGTCTACTGCTGATTAATGGCCAGAAACCAGCCAACCTCGCACAGCGCCGGAGGATACACAGACACAACTGCTTCCTCAAGAGGTGCATACCACTACATTCAAGAGTTCCATTTCCATGAZebrafish (Danio rerio) ELABELA cDNA sequence SEQ ID NO: 41ATGAGATTCTTCCACCCGCTGTATCTGCTGCTGCTGCTGCTGACAGTGCTGGTCCTCATCAGCGCAGATAAACATGGTACAAAACACGATTTTCTCAACTTGAGGCGGAAATATCGCAGACACAACTGCCCGAAGAAACGCTGTCTACCTCTTCACTCCAGAGTACCTTTCCCTTGAHuman (Homo sapiens) ELABELA genomic sequence SEQ ID NO: 42AK092578; LOC100506013; cDNA FLJ35259 fis; clone PROST2004251; Hs. 105196 >gi|21751202|dbj|AK092578.1|Homo sapiens cDNA FLJ25359 fis, clone PROST2004251; Hs. 105196ATCATTAACCTTCCTGCAAAACACAGCTGGCAGTTCTCTGAGGCTTGTCACTAGCCTGTGAAGAC AGCCACACAGATATTGCACAGACTATTTACAGATCGTTTGGCCTTACATTGAGAGTCATTGCTCTACTTTTG TGCGGTAGGAAA ATCAGATTTCAGCAATTCCTTTTTGCATTTTTTATTTTTATTATGAGTCTTCTCCTTATCAGCGGACAGAGACCAGTTAATTTGACCATGAGAAGAAAACTGCGCAAACACAATTGCCTTCAGAGGAGATG TATGCCTCTCCATTCACGAGTACCCTTCCCCTGA GATCTCTCTAGCTAACTTTACTGGATCTATCAGA AGAAGAAGAGGAGTGAAGGAAAGACACCCAGCCACACAAAAGAACTTCATGATGCCAACAGCGTGA TTGCTTAGAAGTTCCTACACAAAAAAAGGATCATTTGAAAGCACCTGGAATGGTTTATTAGCTTCACAGGA TTTTATTCTTCTTGGCTTCTATTTGGAGGGAAAATAACATAAATTCAAAAGGATTCCAATCTGAAGCCCAA ATTGTTTGCCTACATAACAAAAATATCTCATCTTTTCCTGCACATTATTATTCTTTTATGGGTTAAAAAGA AAAATACCTTTTAGTGTTTTAGAACTCTCTCATGGTAAAAAGTGCAAGAATTTAAAATGTTGCTTTCATA TTCCTATAATTCTCCAAAAGTATTAAATTCGTATATGTTTGAGTGATTTTCTAAAAACTGCTCAACCTG GAATCAATTGCATTGACCATTTGGCTTCGCACAATAGGGAGAAAATAATTGGTTCATTGATTATATAGAG AGAAAGACTAAGAAAAGCTATTAATTGCTACCAATTTTATGATAAGCTTTAAGGTTTATGAAAGTATGTT TTTTTATTTAATGAGTAATGTCCATTTGAAGTTGAAAGAAAACATGAAATCCTAATTGTAGTTCATTTTA TGTTCAAATGAACCATTGTTTTTGTTTTTGTTTTGAAACAGAGTCTCACTCTGTTGCCCAAGGTGGAGA GAAGTGGCACGCTTTTGTCTCACTGCAACCTCCACCTCCCGAGTTCAAGTGATTCTCGTGCCTCAACCTCC CAATTATAGGCTGGGATTACAGGTGTGCACCACTACACCCAGCTAATTCTTGTATTTTTTGTAGAGATG AGGTTTTACCCTGTTGCCCAGGCTGGTCTTGAACTCAGGCTGGAACCATTCATTTTTTAACCTTTCTCATC ATGTAATTATAGGAACCCAACGTTTGATTTCCTTTGAAGTTTTGTTATGTCCTTTATTATTTTGTATGGA TAATTTCTTTAAAAGTCTTACTTAAAGTCGACATCTAAAATACAGTTATGCCAATGAAGTCCCACTCAG GGTGATATCTGTATCTAAAAGATGAGTGCTCATCATCCTATTAGGCTTTGTCTTGGTGGTGTTCATCCTGA GATGCTGAGACATGGAAATAAAAAATCAGAAGGAATTTAGGGATATGATTACTCAAAAAAGAAACTA TCCTGTCTAAATTTGAATTGTGTTGATAACTAGGTGTTCCCCAGATGCTAAGATGTTCTTAATTTGTATTTAT TGAAGGATTGTTAGCTTAGTGCCACAAAATTTTTCTTACTTTATGTTAATTCCAGATAAGAAATTTACAA GTTTATATCTTTTTTTTTCTTTTTTTTAAGATGAGATCTGGCTCTATCACCCAGGCTAAAGTGCAGTGGC ATGATCTAGGCTAACTCCCTGGCTCAAGCGATCCTTCCACCTCAGCCTCCCAAGTACCTGGGACTACAG GCACTCACGGCCACACCTGACTAATTTTTGTATTTTTTTGTAGAGATGGAGTATCGCCATGTTGCCCAGG TTGGTCTCAAACTCAGGCTGGTGAGCTCAAGTGATCCGCCTCCTTGGCCTCCCAAAATACTGGGATTACA GGCATGTGCCACCATGCTGGGCCACAAGTTCATATCTGGAGTAGAAGTTTTACTTTGTAAATATTATAAA GTAGAAGAAACCATAAACCATTTTGCTAAAATGAAAGGTTGGGGTTAATATAAATGTAATTTTAAATAG AAAATCTGACAACACTGTCGAGTTTGTCTTCCTGTCAAAGCTTATTAAAAGTGTCTTTGCGGATGAATGGT ACTTTCCACAAGTGCATTTGAGTAGAAGCATAACCTATTCTCAGTTATATTTATGTTTAAAACATGTACT GGTTTGTATATTTTGTACTGAAAAAGAAAACACTTTATAGTCAAGATACATCTCATTCAATACAAGTCTA AACTCTTTCAAATACAAATTCGCATATTCACAGAAAAAGTTACAAATCAGTTTTACTATTGTAAAGTAAT GAAATGGTTATACATTTCTTAATTGTTCAATAAAACACTCAATGATTMouse (Mus musculus) ELABELA genomic sequence SEQ ID NO: 43Mouse Gene AK014119; XM_003084771.1; Mm.58847>gi|74182885|dbj|AK014119.1|Mus musculus 13 days embryo head cDNA, RIKEN full-length enrichedlibrary, clone:3110033I20 product:hypothetical protein, full insert sequenceCGGACTCTCCTTGGAGCTTTGCAGAGACTTCCCGCTTAAGTTACTGCGTGCCTGAATGGAAAAGG CAGCTGGCAGCCCTCTGAGTTCTGGCCATAGGATGTGGGGTGAGCCGGACAGATACTGCGATTTACAGA CGATTTCTCTACTTGAAGAGCTATTGTTCACTTGCGTGTAAGAAAAAAGAAAAACAAAAGGAAAGAAAA AGAAGAAAAGAAAAAAGAGAAAGAAAAGAAAAG ATGCGATTCCAGCCCCTTTTTTGGGTATTTTTTATTTT TGCCAT

TGGCTCTACCAGAAGAAGGGTGAAAGCAAAGATACCCAATCACCGGAAAAACAACCTCAGGAT GGCAACAGGATGGCAGCTCAGGAGTTACTACGCAACAGAGGCTGTTTCAGAGTACCGTGGATGGCTTTTCAGACTGCTCTTCCTGGATTCTCTTTGACAAGAAAATGATAGAAAGGGAAAACAGACGAGGTTAAAGCACATGCG TTTGTCTGAATAACAATCTCTCCTGCTGTTCTGCACGTTCTTTGCGTATAATGTATGAATTACACATAGTGG TGGGTTTCACAAAGGCATTTACATACCAGTACATCACGTCCTATGGCCGTCTTTCCACACATGTGCATC ATATACACCGATTCTTTACAGGCATGCATGCCATATACACTGACTACATTTGCACACACATACCTTCCCC TTTCTCTCCCCTCACCCTCCTGCAGGTCTCTTTTATTCACCTGGGCAGTTTTGTTTCTATTTTAATGGTTTG TACACATGAATGATTTTATTGCACATTATTATTCTTACATGGATTAAAAAGAAAACTACTTTAATChicken (Gallus gallus) ELABELA genomic sequence SEQ ID NO: 44Gga.39575; gene in ensembl: ENSGALT00000038777; Gallus gallus hypothetical proteinLOC770154 (LOC770154), mRNALOCUS XM_001233479  482 bp mRNA linear VRT 16-NOV-2006DEFINITION PREDICTED: Gallus gallus hypothetical protein LOC770154(LOC770154), mRNA. ACCESSION XM_001233479VERSION XM_001233479.1GI:118089826      /gene=“LOC770154” ORIGIN1 ggcgagtgcc acggacgctt ctgtacacac gcggaccgca gggatgaggc tccggcggct61 gctgtgcgtc gtgttcctgc tcctggtcag cctgctacct gccgccgcgc agagaccggc121 caacctggcc ctgcgcagga agctgcaccg acacaactgc tcgcaccggc ggtgcatgcc181 gctccactcc cgcgtgccct ttccctgagc gcccggccca gctcggcaag caatttcgta241 acgggctttt cagtgtctta aaggaggaag ctgcaacaac tgcactgata gagaagctca301 ttctaagtac tgcttaccaa cagttgacct ggtggagcca cagcaatcct gttttgaggg361 agtccatctg aaatgaacac ttcagtggt cctgtgtatc acattctgca tgacctggaa421 caaaggccca tgactcatat cctagaagca gggggaaggg agaaacgggg aaggtgattg481 gg Xenopus (Xenopus laevis) ELABELA genomic sequence SEQ ID NO: 45X1.40684; UGID:1257954; UniGene X1.40684 dbEST Id: 30218202EST name: AGENCOURT_54833116 GenBank Acc: DR729415 GenBank gi:70903527CLONE INFO Clone Id: IMAGE:7975974 (5′)Plate: LLAM17099 Row: n Column: 04 DNA type: cDNA PRIMERSPolyA Tail: Unknown SEQUENCEGTTTCGGTCCGGATTCCCGGGATCCAGACCTGATCTAATAGTTGCCTATCTCTGAAGAGCAATATGGATTTTCAGAAATTATTGTATGCCTTATTTTTCATTCTGATGAGTCTACTGCTGATTAATGGCCAGAAACCAGCCAACCTCGCACAGCGCCGGAGGATACACAGACACAACTGCTTCCTCAAGAGGTGCATACCACTACATTCAAGAGTTCCATTTCCATGAGACCACTGAGGAATCTCATCATGATGTAACATAGGATCTGACTCAAATCTACAGAAATAATATTTATTTTGAACAGTGTTTAAGTTGTTCTTTGACTTATAAGTGGATGTTTCTTAAATGAGCTGCCCACAAGAATGGGCACAGACAAGCTCCAACCCATGTAACTGGCTGTGAAACTGGCACAGGAGCTGCCGAAAGCAATGTCCCTCCATGAAACTGATCCAGGAGACATCTACAGCAACCTTTCTTCCACCAAAACAGACAAGAGAGCAGATTCTCCATCACATTCCAAGGAAGTGAATGTTCAAAGGCTTGCATTTATTATCAAACTGAAATGAAGCATATCATAATAAGGCTCAATGTCCTGTACCTCAAAAGATTTAAAATATGGGGATAAAATGAAGAAAAACAGAAGATGGTTTTTGGACAATCTGGTCATATTTTTAAATATTGCCCCGTGTGCAAAAAAAGAZebrafish (Danio rerio) ELABELA genomic sequence SEQ ID NO: 46Dr.81857; UGID:2443131; UniGene Dr.81857; GenBank: AW777215.1; NCBI Reference Sequence:XP_001335186.1; gi|125803442|ref|XP_001335186.1|1 CATCAGGTCATCTGTCTATCTATCCATCCCTCAGAGGACAGAGAGAGAAGAGAGAGTGAA61 TATCGCCATCTCAAACTTTGAAAAAGTTGGAGAGACCGAGAGCTGGCTAGAACTGCGCGT121 CTTCTATATAACTCAACTTATACGAGATCTGAGAACACTTGCTGAGAGCGACAGACACAT181 AAGAGGATTTCTACAGTCCGTTACCTGCACATCCGACAGAATTTATCGTCTGAGGAACCG241 CGGACATCCTGTGAGGAGAGTCGAGTCTGCGCCGCGGACCAAACCACCCTGAGCATCACC301 ATGAGATTCTTCCACCCGCTGTATCTGCTGCTGCTGCTGCTGACAGTGCTGGTCCTCATC361 AGCGCAGATAAACATGGTACAAAACACGATTTTCTCAACTTGAGGCGGAAATATCGCAGA421 CACAACTGCCCGAAGAAACGCTGTCTACCTCTTCACTCCAGAGTACCTTTCCCTTGAGGT481 TTTATGATGCTCCGGGCAAGCATTAAGAAAAACCAAAGACCAGCCTTGGATTGGAAATGA541 GAAAAGATTTATGTCAGATGTGCCGAGGACTGTTTTATTCGCACATGTATTGTAATCAAA601 GCCATGTTTGTCACTTCTGTAGCAGAAGTGTTTTTTGTTTTGTTTTGTTTTTTAAATGAA661 TGTAAGTGAATGAGCCATGGAGATCCTACTGCTGCCAAACATGCTGCAAACTCATCACTC721 AATCAGGTTGAGTTGGAGCAGAATCATTGTAAATAGTGAGGACTGAATGAAATGTGTTTA781 TATGTAAGTTATGCACTTCAAATGTTTTATTATTATCTTGATTTATTAAAAGTGTATTGT841 CTTTTCAGA Anti-ELABELA shRNA sequence A SEQ ID NO: 47GACACCCAGCCACACAAAA Anti-ELABELA shRNA sequence B SEQ ID NO: 48CCCAGCCACACAAAAGAAC Anti-ELABELA shRNA sequence C SEQ ID NO: 49GTGATTCTCGTGCCTCAAC Anti-ELABELA shRNA sequence D SEQ ID NO: 50CTCACGGCCACACCTGACT Anti-ELABELA shRNA sequence E SEQ ID NO: 51TTGCCTTCAGAGGAGATGT

1. An isolated antibody or antigen-binding fragment thereof thatspecifically binds to one or more of the following: (a) a polypeptidecomprising the sequence CMPLHSRVPFP (SEQ ID NO: 52); (b) a polypeptidecomprising the sequence QRPVNLTMRRKLRKHNC (SEQ ID NO: 53); (c) apolypeptide comprising the sequence QRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP(SEQ ID NO: 2); and (d) an ELABELA polypeptide comprising the sequenceof any of SEQ ID NOs: 1-36.
 2. The isolated antibody or antigen-bindingfragment thereof of claim 1, further comprising a label.
 3. Animmunoassay kit for measuring or detecting ELABELA expression, theimmunoassay kit comprising: (a) a coating antigen comprising one or moreisolated antibodies or antigen-binding fragments thereof thatspecifically binds to one or more of the following: (i) a polypeptidecomprising the sequence CMPLHSRVPFP (SEQ ID NO: 52); (ii) a polypeptidecomprising the sequence QRPVNLTMRRKLRKHNC (SEQ ID NO: 53); (iii) apolypeptide comprising the sequence QRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP(SEQ ID NO: 2); or (iv) an ELABELA polypeptide comprising the sequenceof any of SEQ ID NOs: 1-36; and (b) instructions for using said coatingantigen.
 4. The immunoassay kit of claim 3, wherein the isolatedantibodies or antigen-binding fragments thereof are labelled.
 5. Theimmunoassay kit of claim 3, further comprising an enzyme labelledreagent, a secondary antibody that specifically binds to the isolatedantibodies or antigen-binding fragments, a solid substrate, or anycombination thereof.